The other sucrose-catabolizing enzyme, AI, is a hydrolase, first identified in soybean nodules, that irreversibly cleaves sucrose into fructose and glucose (Copeland and Morell, 1985). The isolation and purification of AI was also carried out from chickpea nodules (Asthir and Singh, 1997). In L. japonicus, the expression level of gene LjInv1 encoding enzyme AI was twofold higher in mature nodules than in uninfected roots. Enzyme activity assays showed that LjInv1 contributed to the production of hexoses and other biosynthetic processes in developing nodules (Flemetakis et al., 2006). In G. max and M. truncatula root nodules, an alkaline/neutral Inv gene was found to have enhanced transcription levels in developing root nodules inferring an increased need for sucrose degradation (Flemetakis et al., 2006; Tesfaye et al., 2006). However, there is not much direct evidence to prove that AI is essential for the regulation of nodule metabolism.

The resulting UDP-Glc and free hexoses (glucose and fructose) are phosphorylated by hexokinase (EC <IP_ADDRESS>) and enter glycolysis or oxidative pentose phosphate pathways. In nodules, the host plant provides carbon sources for bacteroid activities in the form of dicarboxylates, particularly malate and succinate (Day, 1991). Phosphoenolpyruvate carboxylase (PEPC; EC <IP_ADDRESS>) and malate dehydrogenase (MDH; EC <IP_ADDRESS>) convert the carbon flux from glycolysis to form malate (**Figure 1**). PEPC catalyzes the conversion of PEP into oxaloacetate (OAA). The active enzymes are less sensitive to malate, which act as a negative feedback control (Nimmo, 2000). The nodule PEPC was found to be activated with phosphorylation and its sensitivity was inhibited by L-malate in vitro and in vivo (Schuller and Werner, 1993; Zhang et al., 1995). In L. japonicus, abundant transcript of LjPEPC1 was found in the vascular bundles of nodules and bacteroid-infected cells, while LjPEPC2 was expressed in roots and shoots at low levels and postulated as the housekeeping isoform. Moreover, regulatory phosphorylation of PEPC is thought to be mainly controlled by the PEPC kinase (PEPC-PK) (Vidal and Chollet, 1997), and the expression of

Carbon sources are transported across the peribacteroid and bacteroid membranes and enter the TCA cycle in the bacteroid to be metabolized. Transport of inorganic ions and cofactors required for SNF across the SM is indicated. The ammonia produced by the SNF is transported back to the plant and assimilated by GS and GOGAT into Gln and Glu (blue arrows). In indeterminate nodules, Glu and Gln are further converted to Asp and Asn by AAT and AS, respectively (red arrows). In determinate nodules, Gln enters purine synthesis pathway and is converted to ureides (brown arrows). AI, alkaline invertase; UDP-Glc, UDP-glucose; SS, sucrose synthase; PEP, phosphoenolpyruvate; OAA, oxaloacetate; PEPC, PEP-carboxylase; MDH, malate dehydrogenase; PHB, polyhydroxybutyrate; AAT, aspartate aminotransferase; AS, asparagine synthetase; ASP, aspartate; ASN, asparagine.

LjPEPC-PK shares a similar pattern with LjPEPC1 in mature nodules (Nakagawa et al., 2003). The OAA is then converted to malate by MDH in a reversible reaction, and this can then be uptaken by bacteroids directly (**Figure 1**). In alfalfa, five species of cDNAs were found to encode MDH, but only one of them had a nodule-enhanced expression pattern (Miller et al., 1998). One nodule-enhanced MDH (neMDH) was found in garden pea (Fedorova M. et al., 1999). Two MDH isoforms were found in the L. japonicus nodule by transcriptome profiling, one of which is a cytosolic MDH (cMDH) and the other a neMDH (Takanashi et al., 2012). The activity of cMDH in L. angustifolius nodules was shown to increase under P-deficient conditions, supporting the involvement of cMDH in nodules (Le Roux et al., 2014).

Besides the enzymes described above, some other carbon metabolic enzymes also play a role in legume nodules. MsSPSA, encoding sucrose phosphate synthase (SPS; EC <IP_ADDRESS>) in M. sativa L., is involved in the first step of sucrose synthesis, and the expression of MsSPSA showed a nodule-enhanced pattern. SPS showed higher activities in the nodules formed by wild-type S. meliloti, compared to nodules inoculated with a N-deficient Fix− strain (a Tn5 insertional mutation in nifH; Hirsch et al., 1983), suggesting an important role in maintaining a stable carbohydrate/energy supply for the actively nitrogen-fixing symbiont (Aleman et al., 2010).

Multiple environmental stresses result in the inhibition of carbon metabolism in host legumes, resulting in the suppression of SNF efficiency in nodules. Under salt stress, the concentration of sucrose was decreased, and major carbon metabolic enzymes such as SS and AI showed lower activity in both M. truncatula nodules (indeterminate nodules) and L. japonicus nodules (determinate nodules) (López et al., 2008). When garden pea plants were cultured under drought stress, the SS activity was down-regulated, indicating the normal carbon metabolic pathway was inhibited. The NADP-dependent isocitrate dehydrogenase (ICDH; EC<IP_ADDRESS>) was reported to balance C/N metabolic fluxes in leaves (Gallardo et al., 1995; Gálvez and Gadal, 1995). The enhancement of ICDH activity in nodules was probably the result of compensating for the C and N imbalance or supplying additional NADPH to neutralize the effects of the increased production of reactive oxygen species (ROS) as a consequence of drought stress (Gálvez et al., 2005).

### Nitrogen Metabolism

Ammonia exported from the bacteroids diffuses into the cytosol of the infected host cells to be rapidly assimilated. Amino acids and/or ureides are then synthesized and exported from the legume nodules to the shoots (**Figure 1**). The determinate-nodule legumes primarily transport allantoin and allantoate (ureides) as fixed-N compounds, while the indeterminate nodules assimilate amides in the form of asparagine (Asn) and glutamine (Gln) (Sprent, 2009). The first step of ammonium assimilation is apparently the same in amide- and ureide-exporting legume

nodules (Todd et al., 2006). In both cases, nitrogen fixed in the bacteroids is exported into the host cell cytosol, where ammonia is converted to Gln and glutamate (Glu) by the enzymes Gln synthetase (GS; EC <IP_ADDRESS>) and Glu synthase (NADH-GOGAT; EC <IP_ADDRESS>). In indeterminate nodules, Glu and Gln are further converted to aspartate (Asp) and Asn by Asp aminotransferase (AAT EC <IP_ADDRESS>) and Asn synthetase (AS; EC <IP_ADDRESS>). While for determinate nodules, Gln further entry purine synthesis pathway and convert to ureides as final product exported in to host plant (**Figure 1**).

Glutamine synthetase, as the enzyme for the first step of ammonia assimilation in the infected root cells, is a key component of nitrogen metabolism in nodules. It catalyzes the ATP-dependent amination of Glu to Gln. GS possesses two major isoforms largely separated by their subcellular localization, plastidic GS2 and cytosolic GS1 (Temple et al., 1998; Lea and Miflin, 2003). In M. truncatula nodules, three GS genes have been identified: cytosolic MtGS1a and MtGS1b, and plastid MtGS2, where MtGS1a accounts for 90% of the GS activity in nodules (Carvalho et al., 1997, 2000). Impairment of GS activity using phosphinothricin resulted in the inhibition of nodule growth, and the promotion of nodule senescence. The reduced activity of GS also suppressed Asn biosynthesis, as demonstrated by the lower expression of AS transcripts and lower Asn content (Seabra et al., 2012). NADPH-GOGAT is another enzyme involved in the assimilation of NH+ 4 , by transferring the amide group from Gln to α-ketoglutarate (**Figure 1**). In transgenic M. sativa expressing antisense NADPH-GOGAT, GOGAT activities were much reduced in the nodules. The transgenic M. sativa inoculated with S. meliloti exhibited moderate chlorosis, lower nodule weight, and reduction of N2 fixation efficiency compared to the control (Schoenbeck et al., 2000).

In addition to primary nitrogen assimilation by converting ammonia into amino acids, ureide biosynthesis is also a common pathway among tropical legumes such as soybean, common bean, and cowpea, which generally form determinate nodules (Tajima, 2004). The allantoin and allantoate are the final nitrogen forms exported from soybean nodules to the shoots (McClure and Israel, 1979). Before ureide synthesis, ammonia enters a purine synthesis pathway, which is catalyzed by xanthine oxidase and xanthine dehydrogenase into urate, whose activities have been detected in both infected cells and uninfected cells in cowpea nodules. While uricase, which catalyzes the irreversible conversion of urate to allantoin, was detected in uninfected cells but not infected cells (Atkins et al., 1997). Moreover, the genes GmALN3 and GmALN4, encoding the enzyme allantoinase (EC <IP_ADDRESS>) that converts allantoin to allantoate, were expressed in soybean nodules (Duran and Todd, 2012). Gln may also be enzymatically transformed into xanthine through the de novo purine synthesis pathway (Werner and Witte, 2011; **Figure 1**).

### Phosphate Metabolism

Phosphate is the second most limiting macronutrient element required for crop growth after nitrogen, and there is a particularly high demand for phosphates in the nodules of N2-fixing legumes (Gunawardena et al., 1992; Vance et al., 2000). Phosphate, as an essential macronutrient, is incorporated into organic compounds such as nucleic acids (DNAs and RNAs), enzymes, ATP, sugar phosphates, phospholipids, and so on. Such organic phosphates are involved in many plant biochemical processes such as nutrient transport and photosynthesis. As a component of chromosomes, phosphates play an important role in cell division and organogenesis, and help to transfer the genetic information from one generation to the next (Ahemad and Oves, 2011). The soluble inorganic phosphate (Pi) is the most abundant metabolite in Bradyrhizobium japonicum cells. The Pi concentration in free-living B. japonicum cells is 8.1 µmol g−1 FW and 29.2 µmol g−1 FW in bacteroids. Massive amounts of Pi are transported into bacteroids through phosphate transporters and are highly involved in the metabolism of bacteroids. Indeed, SNF expends a great quantity of energy involving phosphorylated intermediates (Vauclare et al., 2013).

### Legume Metabolic Changes Under Pi Deficiency

Deprivation of Pi can lead to an ATP shortage and a decrease in key metabolic enzymes (e.g., those involved in photosynthesis), and therefore is tightly correlated with plant growth (Czarnecki et al., 2013; Saad and Lam-Son, 2017). Although low Pi availability causes a decline in SNF, the responses to Pi deficiency vary among different legumes. There are two main strategies: one is to make changes to nitrogen and carbon metabolism, such as N acquisition and assimilation as well as carbon source adjustments, and the other is to enhance Pi uptake and recycling in nodules (Wang et al., 2010; Qin et al., 2012; Udvardi and Poole, 2013).

To investigate the metabolic changes in legume nodules under low-Pi stress, metabolic profiling has been done in common bean and chickpea (Hernandez et al., 2009; Zhang N. et al., 2012). Hernandez et al. (2009) performed a metabolic profiling using common bean plants inoculated with Rhizobium tropici CIA899 cultivated under Pi-deficient and Pi-sufficient conditions. The levels of 13 metabolites were significantly changed when comparing between the two conditions. Among these, amino acids and other nitrogen metabolites, such as urea, spermidine, and putrescine, were decreased in nodules under low-Pi conditions while most of the carbon metabolites including organic acids and polyhydroxy acids were increased (Hernandez et al., 2009). Similar changes were also found in chickpea inoculated with two different Mesorhizobium strains, M. ciceri CP-31 (McCP-31) and M. mediterranum SWRI9 (MmSWRI9) (Nasr Esfahani et al., 2016). These two Mesorhizobium strains inoculated on chickpea showed a differential symbiotic performance under both control and low-Pi condition. McCp-31-chickpea exhibited a higher symbiotic efficiency and higher Pi concentration than MmSWRI9–chickpea under normal and low-Pi conditions. The differences in SNF efficiency between the two types of chickpea–Mesorhizobium associations were correlated with various changes in carbon and nitrogen metabolites. For instance, the activities of most carbon and nitrogen metabolic enzymes, such as AAT, GS, and MDH, were increased in McCp-31–chickpea nodules, while the activities of these same enzymes were either the same or reduced in MmSWRI9–chickpea nodules. These results provide evidence that host plants adjust their carbon and nitrogen metabolic

pathway to maintain the SNF efficiency under Pi deficiency condition (Nasr Esfahani et al., 2016).

According to previous studies, under low Pi availability, nodules would largely decrease the utilization of atmospheric nitrogen as the nitrogen source and utilize more soil nitrogen (such as NO− 3 and NH+ 4 ) instead, as shown in **Figure 2** (Valentine et al., 2017). The reason for such a shift is not only that NO− 3 itself is a strong inhibitor of nodulation and SNF, but also that the consumption of carbon as an energy source for nitrogen uptake from soil is less than that for SNF (Minchin and Witty, 2005). In Virgilia divaricata, the carbon cost related to SNF ranges from 3.3 to 6.6 gCg−1 -N (gram of carbon was used when fixed each gram of N2), while the carbon cost of NO− 3 reduction is not more than 2.5 gCg−1 -N. Hence, the assimilation of nitrogen from the soil would save more organic carbon for plant growth (Minchin and Witty, 2005). In SNF nodules, the host plant provides photosynthetically fixed carbon to symbiosomes as the major energy source. In turn, bacteria in the nodule fix N2 by the activities of nitrogenases and release NH+ 4 to the host cells. The carbon transported to symbiosomes is mainly in the form of TCA cycle dicarboxylates (such as malate) and is also used as the carbon skeletons for the syntheses of amino acids from N2 (Rosendahl et al., 1990; Valentine et al., 2017; **Figure 2**). For example, in L. angustifolius cv. Wonga, there is an increased rate of malate synthesis via PEPC and MDH under long-term Pi deficiency. Although malate is the major energy source for symbiosomes, high malate accumulation could inhibit N2 fixation and nitrogen assimilation (Le Roux et al., 2008).

### Nitrogen Export From Nodules

As aforementioned, in exchange for the nutrient supply, ammonia and/or alanine and Asp are translocated into the legume cytosol from the symbiosome and then assimilated into ureides or amides. Under Pi deprivation, legumes prefer exporting more ureides, due to the lower carbon cost (**Figure 2**). This was demonstrated through an experimental determination

FIGURE 2 | Mechanisms for maintaining Pi homeostasis under Pi deficiency in legumes. Legumes use more soil N (such as NO− 3 and NH+ 4 ) as the main nitrogen source rather than through SNF. There is also a malate accumulation in both types of nodules and more ureides export in determine nodules under Pi deficiency. A red or purple label means the metabolite content or enzyme activity is up- or down-regulated, respectively. Arrows up (↑) and down (↓) mean the nutrient uptake is up- or down-regulated, respectively. SNF, symbiotic nitrogen fixation; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; OAA, oxaloacetate; MDH, malate dehydrogenase; ASN, asparagine.

of C and N budgets in the tropical legume cowpea and the temperate legume lupin. The results were 1.4 gCg−1 fixed N in cowpea which forms ureide-exporting nodules, and a minimum of 3.9 gCg−1 fixed N in lupin which forms amide-exporting nodules. The result indicates that the export of ureides is more economical in the use of carbon (Atkins, 1991). The shift to higher amounts of ureide export under Pi stress was also observed in soybean, where there was higher accumulation of ureides relative to amino acids in nodules (Le Roux et al., 2009). The switch to ureide exports may reduce the carbon cost under Pi stress, which has a positive effect on legume growth.

### Pi Homeostasis in Legume Nodules

Under Pi stress, legumes exhibit very flexible mechanisms to maintain Pi homeostasis in nodules. On the one hand, legumes increase Pi acquisition from the external environment. On the other hand, plants adopt flexible Pi recycling and internal Pi conservation mechanisms to improve Pi remobilization (Nasr Esfahani et al., 2016; Saad and Lam-Son, 2017).

### **Pi acquisition from the external environment**

Pi acquisition from the external environment by plants is affected by nitrogen metabolism. Pretreatment with NH+ 4 and NO− 3 can increase Pi uptake from soil in Zea mays roots (Smith and Jackson, 1987). Acquisition of NH+ 4 can lead to a release of proton (H+) that decreases the pH in the rhizosphere, which in turn stimulates Pi solubility and uptake (Zhao et al., 2009). Legumes tend to take up NH+ 4 and NO− 3 from the soil through the roots in both Pi stress and Pi sufficient conditions (Dan and Brix, 2009). In turn, more acquisition of NH+ 4 can improve the availability of Pi in the external environment.

Plant acid phosphatases (APases) can hydrolyze organic phosphates to improve soil Pi availability in legumes (**Figure 2**). Under Pi stress, APases become catalytically better using the same amount of nodule crude extract, which is an efficient way to utilize organic phosphates (Araújo et al., 2008). In addition, phytases can hydrolyze phytate into myoinositol and Pi to improve the Pi level in the rhizosphere for uptake through the roots (Araújo et al., 2008). The expression and activities of phytases are also induced by Pi deprivation in P. vulgaris nodules. Two recombinant inbred lines (RILs) of P. vulgaris, RILs 115 (Pefficient) and 147 (P-inefficient), were inoculated with R. tropici CIAT 899 strain and planted in Pi-deficient conditions. Under low Pi condition, the accumulation of phytase transcripts was observed in the nodules of both lines, and it is more enhanced in Line RIL115 than in Line RIL147. The increase in phytase and phosphatase enzyme activities and higher SNF efficiency were found in RIL115 under Pi-deficient treatment, indicating a possible role of phytase activity in nodules to maintain SNF under Pi deficiency condition (Lazali et al., 2013).

The uptake of Pi is also under the regulation of high-affinity Pi transporters. Plants have two identified Pi uptake mechanisms: a high-affinity system that may be up-regulated under low Pi condition and a low-affinity system which is constitutively expressed (Hernandez et al., 2009; Nasr Esfahani et al., 2016). In this review, we focus on the high-affinity Pi transporters. Most high-affinity Pi transporters are induced by Pi starvation and expressed in the root hairs and root epidermis which are directly exposed to soil Pi (Liu et al., 2008). Among Pi transporters, the Pht1 family is the most intensively studied in plants (Gu et al., 2016). Pht1, localized in the plasma membrane, is directly related to Pi uptake from soil and Pi translocation in the plant. Some Pht1s are directly under the control of the central regulators of Pi starvation signaling, the phosphate starvation response (PHR) transcription factors (Rubio et al., 2001; reviewed in Gu et al., 2016). In G. max, 15 Pht1 paralogs have been identified by bioinformatics and experimentation. The expressions of all 15 GmPht1s are up-regulated under low Pi condition compared to high Pi condition. By overexpressing each GmPHT1 paralog in the yeast Pi-transporter mutant PAM2 (1pho84 1pho89), GmPht1;1, GmPht1;2, GmPht1;5, GmPht1;7, and GmPht1;10 are found to be high-affinity Pi transporters and the others are lower affinity Pi transporters (Fan et al., 2013). A Pi starvationinduced high-affinity Pi transporter, GmPT5 (Glyma10g04230), was also identified, which was considered to play an important role in maintaining nodular Pi homeostasis. GmPT5 is expressed in the junction area between roots and young nodules, and mainly functions in transporting Pi from roots to nodules. Higher expression of GmPT5 leads to more Pi being transported from root to nodule, which is beneficial for maintaining SNF (Qin et al., 2012).

### **Internal phosphate recycling and phosphate conservation**

Besides the uptake of Pi from the external environment, legume nodules also develop phosphate recycling and phosphate conservation mechanisms under Pi deficiency (**Figure 2**) (Saad and Lam-Son, 2017). A report showed that chickpea could reallocate Pi from roots to nodules when facing Pi stress. A sharp reduction in Pi (by around 78%) in roots was detected in chickpea, which the researchers assumed that the missing Pi was transported into nodules in order to prevent the complete depletion of nodular Pi (Nasr Esfahani et al., 2016). Common bean was also reported to mobilize Pi from nucleic acids and phospholipids (Hernandez et al., 2009). Under low Pi conditions, V. divaricata exhibited a lower Pi uptake rate but higher levels of phosphohydrolase exudation. This suggests that V. divaricata may prefer to recycle internal nodular Pi pools and use alternate bypass routes to conserve Pi rather than directly uptaking Pi from soil (Vardien et al., 2016).

# Regulation of Metabolism in Legume Nodules of Host Plants

### Regulators of Carbon and Nitrogen Metabolism

It is an important challenge for legumes to maintain a balance between supplying the nodules with the amount of carbon required for nitrogen fixation while retaining sufficient carbon for growth, as well as keeping the nitrogen efflux from the nodules at an optimum level. The host plant regulates the symbiotic process by controlling nodule development and nodule numbers as well as by adjusting the nodule turnover and the level of nitrogen fixation (Ferguson et al., 2010; Sulieman and Schulze, 2010). The balance between plant nitrogen demand and nitrogen fixation rate is finely tuned to reach the nitrogen concentration that allows maximum plant growth. In common bean, nitrogen translocated

from senescing lower leaves to nodules resulted in lower nitrogen fixation rate (Fischinger et al., 2006). Autoregulation of nodulation (AON) is the main systemic negative feedback mechanism of host plants to negatively regulate SNF in nodules (Ferguson et al., 2010). AON has been identified using several legume mutants with the super nodulation phenotype, such as the G. max mutant nts-1, M. truncatula mutant sunn, and L. japonicus mutant har1, were found to lack in a leucine-rich repeat receptor-like protein kinase for AON in shoots (Krusell et al., 2002; Nishimura et al., 2002; Schnabel et al., 2005). CLAVATA3/embryo-surrounding region (CLE) peptides are a group of small (12–13 amino acids) secreted peptides derived from the C-terminal region of preproproteins. In M. truncatula, it has been suggested that MtCLE12 or MtCLE13 plays a role for CLE signaling in controlling nodule numbers. These peptides are generated in roots and transported to leaves via the xylem to trigger the AON response (Mortier et al., 2010). LjCLE-RS1 and LjCLE-RS2 were also found to be involved in regulating nodule formation in L. japonicus (Magori and Kawaguch, 2010).

### Regulator for Pi Homeostasis

Legumes have evolved to adopt strategies to maintain the nodular Pi homeostasis (Sulieman and Tran, 2015). Multiple genes and proteins work as regulators (Rubio et al., 2001; Yao Z.F. et al., 2014; Sulieman and Tran, 2015). A MYB-CC-type transcription factor (PHR1), a small non-coding RNA (microRNA399), and proteins containing the SYG1/PHO81/XPR1 (SPX) domain all play important roles in Pi stability in nodules, and are considered to be vital regulators (Xue et al., 2017). DNA methylation may also be involved in the nodular Pi homeostasis regulation, but it still needs further research (Kim et al., 2015; Yong-Villalobos et al., 2015; Crampton et al., 2016).

In soybean, 35 GmPHR members have been identified. Among them, GmPHR25 is induced by Pi starvation and it in turn increased the transcripts of 11 out of 14 high-affinity Pi transporters as well as those of 5 other Pi starvationresponsive genes (Xue et al., 2017). This evidence indicated that GmPHR25 is a vital Pi homeostasis regulator in soybean (Xue et al., 2017). Besides small non-coding RNAs, long non-coding RNAs (lncRNAs) are also involved in Pi homeostasis. In the legume model plant, M. truncatula, three phosphate deficiencyinduced LncRNAs (PDILs) have been characterized through their corresponding Tnt1 mutants. PDIL1 suppresses the degradation of MtPHO2, working as a positive Pi homeostasis regulator. However, PDIL2 and PDIL3 may directly inhibit Pi transporters, therefore working as negative regulators (Wang et al., 2017).

Proteins containing the SPX domain are considered to be vital regulators in the plant Pi signaling network (Yao Z.F. et al., 2014). In common bean, PvSPX1–PvSPX3 are all induced by Pi starvation, but PvSPX1 had higher sensitivity and faster response to Pi starvation than the other two PvSPXs. Ten Pi starvationresponsive genes are induced by overexpressing PvSPX1, even with an increase in Pi concentration at the same time. It was further shown that the overexpression of PvPHR1 resulted in a decrease in PvSPX1 transcripts. Therefore, PvSPX1 is a positive regulator in maintaining Pi homeostasis and is itself regulated by PvPHR1 (Yao Z.F. et al., 2014). But in soybean, GmSPX1 is a negative regulator in the Pi signaling network, and may be involved in phosphate starvation by inhibiting the expression of GmMYB48, a phosphate starvation-induced gene (Zhang et al., 2016). GmSPX3 is also induced by Pi starvation, and considered to be a positive regulator which induces seven Pi starvationresponsive genes in soybean hairy root (Yao Z. et al., 2014). Genes found to be associated with legume Pi homeostasis are listed in **Table 2**.

# METABOLISM AND TRANSPORT IN BACTEROIDS

### Nitrogen Assimilation Nitrogenase Complex

Symbiotic nitrogen fixation by rhizobia in legume root nodules is carried out through the nitrogenase enzyme complex. Nitrogenase catalyzes the following reaction:

$$\text{N}\_2 + 8\text{e}^- + 8\text{H}^+ + 16\text{ATP} \rightarrow 2\text{NH}\_3 + \text{H}\_2 + 16\text{ADP} + 16\text{Pi}\_2$$

This reaction describes the reduction of nitrogen to ammonia, and is associated with a high energetic cost (ATP). The enzymatic complex of nitrogenase consists of two enzymes: dinitrogenase reductase, a dimeric Fe-protein encoded by the nifH gene, and dinitrogenase, a tetrameric FeMo-protein encoded by the nifDK gene (Rascio and La Rocca, 2013; Rubio and Ludden, 2015). These enzymes are rapidly deactivated in the presence of atmospheric concentrations of oxygen (Dixon and Wheeler, 1986).

### Regulation of Nitrogenase Activity

Nitrogenase activity is reduced at high oxygen concentrations. Nodules provide low oxygen environments for the N2-fixing bacteroids, as observed in soybean nodules where the oxygen concentration is 56 nM (Kuzma et al., 1993) and even lower concentrations have been registrated (Appleby, 1984). Therefore, oxygen tension is a key regulator of genes required for N2 fixation, nitrogenase synthesis (nif genes), and microoxic respiration (fix genes). The nitrogen fixation signaling pathway differs between rhizobium strains. In S. meliloti, the hemecontaining FixL, a signal-transducing membrane protein, acts as the oxygen sensor for the system. In the absence of oxygen, FixL autophosphorylates and transfers the phosphate to FixJ (Gilles-Gonzalez et al., 1991; Lois et al., 1993). This transcriptional factor in turn regulates positively the expression of two regulatory genes, nifA and fixK. NifA is a transcriptional activator that controls the expressions of nitrogen fixation genes, fixABCX, nifN, nifB, and nifHDK (Gong et al., 2006). FixK upregulates the expression of fixNOQP and fixT (Batut et al., 1989; Foussard et al., 1997). FixT represses the expression of nifA (Foussard et al., 1997) and FixNOQP is a cytochrome terminal oxidase cbb3 with a high affinity for oxygen that allows ATP production required for the nitrogen fixation process (Preisig et al., 1993). However, in B. japonicum, the FixLJ system is not required to induce the expression of nifA (Fischer and Hennecke, 1987), but instead it is RegSR that mediates positive control to transcription of nifA (Lindemann et al., 2007).

FixLJ cascade system varies in some bacteria. FnrN, belonging to the CRP-Fnr family of global transcriptional factors found in bacteria, acts as the oxygen sensor in R. leguminosarum bv. viciae VF39 and Rhizobium etli CNPAF512 (Patschkowski et al., 1996; Moris et al., 2004). Both organisms synthesize FnrN and FixK. In R. leguminosarum, FnrN is the main oxygen sensor responsible for the induction of fixNOQP and is in turn induced by the FixL protein (Boesten and Priefer, 2004). In contrast, the expression of fnrN is autoregulated in R. etli CNPAF512 (Moris et al., 2004). Other cases of rhizobia oxygen-response cascade have been previously described in detail (Terpolilli et al., 2012).

### Dicarboxylate Metabolism

Most rhizobia are obligate aerobes. The C4-dicarboxylic acids supplied by the host plant must be metabolized through the tricarboxylic acid (TCA) cycle in the bacteroid. Malate, the primary carbon source of bacteroids, is converted to pyruvate and CO2 via the NAD+-dependent malic enzyme (DME). Pyruvate is subsequently decarboxylated by pyruvate dehydrogenase to form acetyl-coenzyme A (acetyl-CoA) and enters the TCA cycle. DME is required to provide pyruvate to the TCA cycle in S. meliloti and Azorhizobium caulinodans (Zhang Y. et al., 2012). S. meliloti possesses two distinct malic enzymes, an NAD(P)+-dependent enzyme (DME) (EC <IP_ADDRESS>) and a strictly NADP+-dependent enzyme (TME) (EC <IP_ADDRESS>) (Driscoll and Finan, 1993, 1996; Voegele et al., 1999). In addition, acetyl-CoA can be produced alternatively by phosphoenolpyruvate carboxykinase (PCK) which catalyzes the decarboxylation of OAA to PEP. Symbiotic phenotypes of pck mutants vary depending on the host plant. pckA mutants of R. leguminosarum MNF3085 fix nitrogen at rates comparable to wild type, while pckA mutants of S. meliloti show a reduced N2 fixation capacity (McKay et al., 1985; Finan et al., 1991). In R. leguminosarum and S. fredii strain NGR234, DME or a pathway involving PCK and pyruvate kinase (PYK) can synthesize the precursors required for SNF (Zhang Y. et al., 2012). Enzymes participating in the TCA cycle have been identified in B. japonicum strain USDA110 (Sarma and Emerich, 2006), R. leguminosarum (McKay et al., 1985), S. meliloti (Djordjevic, 2004), and R. tropici (Romanov et al., 1994). However, TCA cycle involvement probably varies among rhizobia. Evidence shows that S. meliloti, R. tropici, and R. leguminosarum utilize the full oxidative TCA cycle to provide

ATP, precursors of amino acid synthesis, as well as reducing equivalents for N2 fixation. Enzymes of the TCA cycle appear to be essential for nitrogen fixation in S. meliloti since mutations in succinate dehydrogenase (sdh), malate dehydrogenase (mdh), isocitrase dehydrogenase (icd), 2-oxoglutarate dehydrogenase, aconitase (acnA), and citrate synthase (gltA) abolish N2 fixation, despite that nodules were formed (Duncan and Fraenkel, 1979; McDermott and Kahn, 1992; Mortimer et al., 1999; Dymov et al., 2004; Koziol et al., 2009). In contrast, B. japonicum shows a higher metabolic plasticity. Mutations in fumarase (fumC), ICDH (idhA), alpha-ketoglutarate dehydrogenase (agdA), and acotinase (acnA) in B. japonicum USDA110 still show phenotypes capable of fixing nitrogen (Acuña et al., 1991; Thöny-Meyer and Künzler, 1996; Green and Emerich, 1997; Shah and Emerich, 2006). Transcriptome analyses of R. etli bacteroids have suggested that the TCA cycle is inactive in these bacteroids (Vercruysse et al., 2011), but additional experiments are required to explain how the bacteroids obtain the ATP necessary for nitrogen fixation in these cases. A comparative metabolic profiling study between free-living R. leguminosarum and pea bacteroids showed that the TCA cycle is not the only path to oxidize dicarboxylic acids derived from the host plant in the bacteroids (Terpolilli et al., 2016). Metabolic profiling and flux analysis revealed that pea bacteroids divert acetyl-CoA into TCA and the production of lipid or polyhydroxybutyrate (PHB). These findings suggest new pathways for electron allocation in nitrogen-fixing bacteroids where lipogenesis may be a requirement in legume nodules.

### Carbon Storage

The legume–rhizobium symbiosis determines the accumulation of specific metabolites inside the microsymbiont. Excess carbon and reducing power provided by the host plant can be stored as polymers, glycogen, or lipids in bacteroids (**Figure 1**). PHB is accumulated in large cytoplasmic granules in the bacteroids that form determinate nodules (such as in common bean and soybean), but not in the indeterminate nodules of alfalfa, garden pea, and chickpea (**Table 1**; Tombolini and Nuti, 1989; Kim et al., 1996).

The PHB biosynthesis pathway consists of three steps. β-Ketothiolase (PhaA) catalyzes the first step in this pathway with the formation of acetoacetyl-CoA from acetyl-CoA. Acetoacetyl-CoA is then reduced to D-β-hydroxybutyryl-CoA by an NADHdependent acetoacetyl-CoA (PhaB), and then PHB synthase (PhaC) catalyzes the final formation of PHB (Lodwig and Poole, 2003). In S. meliloti and R. etli, phaA and phaB form an operon, phaAB.

Fix+ symbiotic phenotypes have been observed in S. meliloti when mutations were introduced to disable the synthesis of PHB or alter the ability to utilize PHB cycle intermediates to support growth (Povolo et al., 1994; Aneja and Charles, 1999; Cai et al., 2000). Effective nodules were also observed when R. leguminosarum mutant strains defective in the phaC gene were used to inoculate bean (determinate nodules) or pea (indeterminate nodules) (Lodwig et al., 2005). However, a reduced nitrogen fixation capability was observed when PHB synthesis was abolished in A. caulinodans. In A. caulinodans, impaired PHB synthetase activity results in the loss of nitrogen fixation capacity both ex planta and in symbiosis with the tropical legume Sesbania rostrata (Mandon et al., 1998). These evidences suggest that PHB may play certain roles in nitrogen fixation of some legume–rhizobium systems.

Polyhydroxybutyrate synthesis is regulated in R. etli and S. meliloti by the PHA (polyhydroxyalkanoate) regulator (PhaR), previously called AniA (Povolo and Casella, 2000; Encarnación et al., 2002). PhaR homologs in other bacterial species bind to their own promoters and to the promoter of phaP (Maehara et al., 2001). PhaP, a phasin, binds to the surface of PHB granules and can control the size of the PHB granules (Kuchta et al., 2007; Mezzina et al., 2014). In Bradyrhizobium diazoefficiens, the regulation of PHB synthesis is mediated through the interaction between PhaR and FixK2, regulators associated with the synthesis of PHB and microoxic metabolism (Quelas et al., 2016).

Glycogen is co-produced with PHB in free-living rhizobia such as R. leguminosarum and S. meliloti under nutrientlimiting conditions (Povolo et al., 1994; Lodwig et al., 2005). The role of glycogen in SNF has been studied in some rhizobia. A glycogen synthase mutant (glgA) of R. tropici showed an enhanced symbiotic performance as measured by an increase in plant dry weight and nodule number, but the mechanisms behind this phenotype are uncertain (Marroquí et al., 2001). In garden pea bacteroids, a glgA mutant of R. leguminosarum did not alter nitrogen fixation rates, as indicated by the acetylene reduction assay for nitrogenase activity. Plants inoculated with a phaC/glgA double mutant resulted in similar phenotypes to wild-type-inoculated plants (Lodwig et al., 2005). In contrast, the glgA mutant of S. meliloti resulted in lower levels of N2 fixation in both M. truncatula and M. sativa (Wang et al., 2007). Thus, the SNF capability of S. meliloti is affected by both PHB and glycogen availability.

In Bradyrhizobium sp. ORS278, studies have shown that mutations in genes involved in the Calvin–Benson–Bassham (CBB) cycle result in deficiency in nitrogen fixation. The CBB cycle consumes 3 mol of ATP and 2 mol of reducing equivalents per mol of carbon dioxide. The symbiotic phenotypes suggest that Bradyrhizobium sp. ORS278 uses the Calvin cycle as a reductant store to regulate carbon flux. Specifically, phosphoglycerate kinase (CbbK) and ribulose 1,5-biphosphate carboxylase (RuBisCO) large chain (CbbL1) have been identified as being involved in the symbiotic process (Bonaldi et al., 2010; Gourion et al., 2011). Thus, the relevance of different carbon storage compounds in the symbiotic process varies depending on the rhizobial strain.

### Transport in Bacteroids

### Symbiotic Membrane: A Regulatory Barrier

Mature bacteroids drive nitrogen fixation inside root nodules, releasing ammonia to the plant cell cytosol to be incorporated into nitrogenous compounds. Ammonia assimilation is severely

reduced in bacteroids due to a coordinated response where growth-associated pathways are switched off and biosynthesis of amino acids is downregulated but still present (Li et al., 2013). However, bacteroids require the supply of some plantderived amino acids to support bacteroid development (Prell et al., 2009; Mulley et al., 2011; reviewed in Dunn, 2014). The supply of amino acids is plant-type specific (Randhawa and Hassani, 2002). For example, alfalfa is not able to provide sufficient histidine to the histidine auxotrophs of S. meliloti in nodules (Malek and Kowalski, 1977) unlike the cowpea host plant where similar mutants of rhizobial strain IRC256 are able to fix nitrogen (McLaughlin et al., 1987). The transport of nutrients is regulated by the symbiosome membrane (SM). SM is a plant-derived membrane that controls the metabolite exchanges in the legume–rhizobia symbiosis via specific transport systems and encloses a single bacteroid in indeterminate nodules or several bacteroids in determinate nodules (Clarke et al., 2014). Different transport mechanisms have been identified across the legume SM such as carbon, nitrogen, and cation transport systems, but most of them are not characterized (Day et al., 2001; Clarke et al., 2015). The main metabolite transport systems are schematically represented in **Figure 1**.

### Dicarboxylate Transport

C4-Dicarboxylates are the major metabolites that are transported across SM to generate ATP in the bacteroids. A dicarboxylate transporter for malate and succinate has been identified in the soybean SM (Udvardi et al., 1988). This transporter possesses a higher affinity for malate than for succinate. However, the gene encoding the transporter has not been identified in any legume. A dicarboxylate transporter has been characterized in the SM of a non-legume, Alnus glutinosa AgDCAT1 was shown to transport dicarboxylates (malate, succinate, fumarate, and OAA) when expressed in Escherichia coli (Jeong, 2004). AgDCAT1 belongs to the peptide transporter family (PTR) and proteins from this family are candidates for dicarboxylate transporters in legumes since experimental evidence shows that PTR-encoding genes are induced in legume nodules (Colebatch et al., 2004; Libault et al., 2010). Members of this family have been identified in the proteomic studies of the soybean SM, but their functions have not been characterized (Clarke et al., 2015). In rhizobia, a C4-dicarboxylate transport system (Dct) has been identified and characterized. Dct consists of a permease encoded by dctA and a two-component sensor-regulator system (DctBD) encoded by dctB and dctD. DctBD responds to the presence of C4 dicarboxylates and regulates dctA expression (reviewed in Yurgel and Kahn, 2004). dctA mutants of Rhizobium trifolii are unable to transport dicarboxylates and form ineffective nodules (Fix−) in Trifolium repens and Trifolium pratense. The resulting infected plant cells accumulated large amounts of starch (Ronson et al., 1981).

### Ammonia and Ammonium Transport

The ammonia produced by nitrogen fixation in bacteroids is probably protonated to ammonium after diffusing into the peribacteroid space (PBS) or symbiosome space (SS, the region between the SM and the bacteroid) (**Figure 1**). An H+-ATPase pumps H+ into the PBS, generating an acidic environment (Udvardi et al., 1991) and forming a membrane potential (Udvardi and Day, 1989). ATPase activity has been detected on the SM of soybean (Blumwald et al., 1985; Fedorova E. et al., 1999), lupin (Robertson et al., 1978; Domigan et al., 1988), and garden pea root nodules (Szafran and Haaker, 1995). Re-uptake of NH+ 4 by bacteroids is prevented by the repression of the bacteroid ammonium carrier, Amt, during the symbiotic state (Howitt et al., 1986). At present, two pathways have been proposed for the transport of fixed nitrogen into the host plant: a monovalent cation channel for NH+ 4 (Whitehead et al., 2008) and an aquaglyceroporin, nodulin 26 (Nod26), for NH3 transport. Nod26 is a transmembrane protein able to transport H2O, NH3, and other solutes (Weaver et al., 1994; Rivers et al., 1997; Hwang et al., 2010). It was first identified in the soybean SM (Fortin et al., 1987) and constitutes the major protein component of the SM (Rivers et al.,

### Amino Acid Transport

1997).

Host plants supply amino acids to the microsymbionts during the differentiation of rhizobia to bacteroids and for nitrogen fixation (Mulley et al., 2011). Recent analyses of the soybean proteome have revealed a putative amino acid transporter (GmAPC1), a homolog to members of the amino acidpolyamine-organocation (APC) family of transport proteins (Clarke et al., 2015). Previous studies have shown that the soybean SM is permeable to alanine and Asp (Whitehead et al., 1998). An Asp transporter has been identified in SM vesicles from root nodules of garden pea (Rudbeck et al., 1999).

In addition, rhizobia also encode transport systems that regulate the exchange of amino acids between the bacteroids and the host cell (Udvardi and Poole, 2013). For instance, while the bacteroids formed by R. leguminosarum bv. viciae in the indeterminate pea nodules are auxotrophs for branched amino acids, branch-chain amino acid transporters, Aap (AapJQMP) and Bra (BraDEFGC) are developed in the bacteroids (Prell et al., 2009). This symbiotic phenotype is known as symbiotic auxotrophy (Dunn, 2014) and occurs in both determinate and indeterminate nodules (de las Nieves Peltzer et al., 2008; Prell et al., 2010; di Cenzo et al., 2015).

TABLE 3 | Available metabolic reconstructions of rhizobia. Model Organism Scope Reference iOR363 R. etli SNFa Resendis-Antonio et al., 2007 iOR450 R. etli SNFa Resendis-Antonio et al., 2012 iHZ565 S. meliloti SNFa Zhao et al., 2012 iGD1575 S. meliloti Whole cell di Cenzo et al., 2016 iGD726 S. meliloti Core metabolism di Cenzo et al., 2018 iYY1101 B. diazoefficiens Whole cell Yang et al., 2017

aSNF, symbiotic nitrogen fixation.

### Metabolic Modeling of SNF

fpls-09-01860 December 17, 2018 Time: 16:30 # 12

Constraint-based modeling enables the determination of the metabolic capabilities of an organism (Feist et al., 2009; Oberhardt et al., 2009). These metabolic models have been used successfully to bridge the gap between current knowledge and metabolic phenotypes (Schellenberger et al., 2011). Metabolic reconstructions are based on physiological and biochemical information on primary and specialized metabolic pathways, genome information, and available -omics data. These models have allowed for the prediction of the metabolic behavior of microorganisms under different nutrient availabilities and simulated gene deletions or over-expressions (Contador et al., 2015; Razmilic et al., 2018). In an attempt to get new insights into the genetic interactions that orchestrate the complex metabolic interactions in nitrogen-fixing bacteria, metabolic models have been used to study the rhizobial metabolism. Genome-scale models have been combined with experimental data to identify essential genes and to simulate the metabolic behaviors of rhizobia under nitrogen fixation conditions in legume nodules. High-throughput experimental data aimed to identify key genes, proteins and metabolites have been used in these studies to validate the model predictions.

At present, few manually curated rhizobial reconstructions are available despite their important roles in sustainable agriculture. These reconstructions cover different metabolic tasks from only the symbiotic process that takes place in the rhizobium to the complete set of metabolic reactions of the bacteria cell. iOR363, a reconstruction for R. etli CFN42, was the first model available for any rhizobium (Resendis-Antonio et al., 2007). This model describes the SNF process inside the determinate nodules of P. vulgaris and includes the main reactions associated with the nitrogen fixation process and the metabolic pathways, such as PHB synthesis, that allow simulating the accumulation of this polymer during the symbiotic process (Resendis-Antonio et al., 2007). Also, a symbiotic reaction was defined to represent this specific legume–rhizobia symbiosis. The symbiotic reaction describes the exchange of nutrients between the host plant and the bacteroid to establish symbiotic relationships and to obtain or produce chemical compounds required by the process of nitrogen fixation. A symbiotic auxotrophy hypothesis and nitrogenase requirements were included in its definition. This model has been updated to include new data sets available of this system (Resendis-Antonio et al., 2011, 2012).

On the other hand, the metabolic reconstructions for S. meliloti have different scopes. iHZ565 was designed to represent the SNF of S. meliloti 1021 with host plants such as alfalfa (Zhao et al., 2012). The reconstruction process gave insight about the missing context-specific information and genome annotation errors. Additionally, a new symbiotic reaction was proposed to capture specific SNF mechanisms of S. meliloti in indeterminate nodules. The first representation of the whole metabolism of a rhizobium cell was built for S. meliloti (iGD1575) to enable the characterization of the metabolic capabilities of rhizobia in bulk soil, rhizosphere, and nodule (di Cenzo et al., 2016). iGD1575 encompasses 94% of the genes present in iHZ565. Recently, iGD1575 and gene essentiality experiments were used to build a model of the core metabolism of S. meliloti (di Cenzo et al., 2018). Pfau et al. (2018) have described the symbiotic relationship of M. truncatula with S. meliloti through constraint-based modeling (Pfau et al., 2018). Metabolic models were used to study the metabolic exchange between the host plant and the rhizobial symbiont. An in silico representation of the metabolic network of B. diazoefficiens USDA110 (iYY1101) has also been constructed (Yang et al., 2017). This reconstruction was used to build context-specific models to describe the metabolic differences observed between the free-living state of B. diazoefficiens USDA110 and symbiotic bacteroid. These models will provide a starting point for building metabolic reconstructions of closely related organisms such as other rhizobial strains and modeling frameworks to study these symbiotic interactions (Baumler et al., 2011; Monk et al., 2013; Ong et al., 2014). **Table 3** summarizes the available metabolic models of rhizobia.

### CONCLUSION

The regulatory mechanisms regarding the formation and maintenance of root nodules for SNF are multi-faceted and association-specific, but they all involve the tight regulation of the flow of metabolites between the host plants and the bacteroids, as well as the different levels of controls over each step in the metabolic pathways through the metabolic enzymes involved and the transcription factors that regulate their expressions. Each host plant–rhizobium association has its own unique requirements and control mechanisms despite the overall common features. It is therefore critical that we have as much understanding as possible of the detailed mechanisms that make up this symbiotic relationship.

# AUTHOR CONTRIBUTIONS

H-ML designed the conceptual framework of this paper and coordinated the writing. AL and KF wrote the part on metabolism and regulation of host plant. CC wrote the part on metabolism and regulation of bacteroids and metabolic models. AL, CC, and H-ML revised and polished the article.

### FUNDING

This work was supported by the Hong Kong Research Grants Council General Research Fund (14108014) and Area of Excellence Scheme (AoE/M-403/16), CUHK VC Discretionary Fund VCF2014004, and the Lo Kwee-Seong Biomedical Research Fund to H-ML.

### ACKNOWLEDGMENTS

Jee-Yan Chu copy-edited this manuscript.

### REFERENCES

FixL of Rhizobium meliloti are coordinately regulated by oxygen. J. Biol. Chem. 268, 4370–4375.

fixation by globally downregulating transport of organic nitrogen sources. Mol. Microbiol. 80, 149–167. doi: 10.1111/j.1365-2958.2011.07565.x

between free-living and symbiotic states. Mol. Biosyst. 13, 607–620. doi: 10. 1039/C6MB00553E

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Liu, Contador, Fan and Lam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Water-Soluble Humic Materials Regulate Quorum Sensing in Sinorhizobium meliloti Through a Novel Repressor of expR

Yuan-Yuan Xu1 , Jin-Shui Yang1 , Cong Liu1 , En-Tao Wang2 , Ruo-Nan Wang1 , Xiao-Qian Qiu1 , Bao-Zhen Li1 , Wen-Feng Chen1 and Hong-Li Yuan1 \*

1 State Key Laboratory of Agrobiotechnology and Key Laboratory of Soil Microbial, Ministry of Agriculture, College of Biological Sciences, China Agricultural University, Beijing, China, 2 Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico

### Edited by:

Hon-Ming Lam, The Chinese University of Hong Kong, China

### Reviewed by:

Muthu Venkateshwaran, University of Wisconsin–Platteville, United States Yangrong Cao, Huazhong Agricultural University, China

### \*Correspondence:

Hong-Li Yuan<EMAIL_ADDRESS><EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

Received: 03 September 2018 Accepted: 10 December 2018 Published: 21 December 2018

### Citation:

Xu Y-Y, Yang J-S, Liu C, Wang E-T, Wang R-N, Qiu X-Q, Li B-Z, Chen W-F and Yuan H-L (2018) Water-Soluble Humic Materials Regulate Quorum Sensing in Sinorhizobium meliloti Through a Novel Repressor of expR. Front. Microbiol. 9:3194. doi: 10.3389/fmicb.2018.03194 Quorum sensing (QS) plays an important role in the growth, nodulation, and nitrogen fixation of rhizobia. In this study, we show that water-soluble humic materials (WSHM) repress the expression of the QS related genes sinI, sinR, and expR in Sinorhizobium meliloti. This decreased the production of N-acetyl homoserine lactones (AHL) and exopolysaccharides (EPS), and ultimately increased S. meliloti cell density. We also identified a novel regulator, SMc03890 (renamed QsrR), which binds directly to the expR promoter. Deletion of qsrR increased expR expression. WSHM repressed the expression of expR by augmenting the interaction between QsrR and the expR promoter; this was determined by a bacterial-one-hybrid assay. These effects of WSHM on the QS system in S. meliloti may be the underlying mechanism by which WSHM increase the symbiotic nitrogen fixation of Medicago sativa inoculated with S. meliloti. This study provides the first evidence that humic acids regulate the QS of rhizobia and suggests that WSHM could be used as fertilizers to improve the efficiency of symbiotic nitrogen fixation.

Keywords: Sinorhizobium meliloti, quorum sensing, humic materials, ExpR regulator, bacterial communication

# INTRODUCTION

Quorum sensing (QS) is a bacterial communication mechanism in which cell physiology and behavior are coordinated with population density (Bogino et al., 2015). In symbiotic nitrogen-fixing bacteria (rhizobia), QS plays a key role in their growth and the formation of symbiosis with their legume hosts (Bogino et al., 2015; Koul et al., 2016). In the QS system of Sinorhizobium meliloti 8530 (**Figure 1**), SinI is responsible for synthesis of the QS signaling molecules, N-acetyl homoserine lactones (AHL). The expression of sinI is induced by SinR; meanwhile, ExpR either mediates a positive regulatory feedback loop by inducing the expression of sinI or a negative feedback regulation by down-regulating the expression of sinR, depending on the AHL concentration (Charoenpanich et al., 2013; Calatrava-Morales et al., 2018). In addition to regulating the genes involved in nodulation and nitrogen fixation (Hoang et al., 2004), AHL-ExpR complex also upregulate the expression of exp operon, which is involved in EPS II synthesis, and down-regulate genes related to bacterial motility, such as visN, visR, and rem (Gurich and Gonzalez, 2009; Mueller and González, 2010). The number of pink nodules induced by the sinI mutant decreased compared

with the WT strain due to the inability of the sinI mutant to repress the expression of motility genes at high cell densities (Gurich and Gonzalez, 2009). The expR mutant, S. meliloti 1021, grows more rapidly and has an increased nodule occupancy (10∼20% higher than the WT strain, S. meliloti 8530) (Charoenpanich et al., 2015).

Many compounds have been reported to interfere with the bacterial QS systems (Kalia, 2013; Gonzalez-Ortiz et al., 2014). For rhizobia, L-canavanine, which is extracted from alfalfa seed exudates, inhibits the expression of S. meliloti expR gene (Keshavan et al., 2005), while the flavonoids induce the expression of AHL synthesis genes in Sinorhizobium fredii SMH12 and in Rhizobium etli ISP42 (Pérez-Montaño et al., 2011). Generally, compounds that interfere with microbial QS systems are heterocyclic compounds containing groups such as furan, pyridine, butyrolactone, benzene ring, and quinoline (Gonzalez and Keshavan, 2006; Christensen et al., 2013). These compounds have chemical structures similar to those of humic materials (Gao T.G. et al., 2015); thus, it is possible that humic materials may also interfere with microbial QS systems.

Humic materials are supramolecules derived from the residues of degraded plant, animal and microbial cells (Hayes and Wilson, 1997), with structures of relatively small selfassembled molecules that are held together by multiple weak interactions, such as hydrogen and van der Waals bonds. Generally, humic materials include, but are not limited to, n-alkanoic acids, n-alkanols, hydrocarbons, hydroxyacids, aromatic compounds, polyhydroxylated compounds, steroids, terpenoids, and N-heterocyclic compounds (Nebbioso and Piccolo, 2012). Humic materials are the most biological active compounds in soil that could stimulate plant growth as phytohormones (Traversa et al., 2013; Savy et al., 2017), enhance ATPase activity, promote nutrient utilization of plant (Jannin et al., 2012; Canellas et al., 2015), and stimulate the growth of bacteria (Tikhonov et al., 2010). In a previous study, we revealed that the water-soluble humic materials (WSHM) that are produced by lignite biodegradation enhanced the growth, cell metabolism, and nutrient transport of Bradyrhizobium liaoningense CCBAU05525, as well as its nodulation with soybean (12∼26% increased yield in soybean grains) (Gao T.G. et al., 2015). Therefore, WSHM could be a potential fertilizer to improve the legume yield.

In the present study, we examined the effects of WSHM on the QS system and the symbiotic nitrogen fixation of S. meliloti, and revealed that WSHM enhance the growth and nitrogen fixation of S. meliloti by regulating the QS system. This is the first report showing the effects of WSHM on the QS system in S. meliloti. Finally, we identified QsrR as a novel repressor of expR in this bacterium.

# MATERIALS AND METHODS

### WSHM, Bacterial Strains, and Plasmids

Water-soluble humic materials were extracted from lignite collected from a Coal Mine in Inner Mongolia of China according to previously described methods (Dong et al., 2006; Jiang et al., 2013). Briefly, the lignite powder was inoculated with Penicillium sp. P6 and Bacillus sp. Y7 and incubated at 28◦C for 2 weeks. Biodegraded lignite was diluted tenfold in deionized water, stirred, and centrifuged three times at 9000 × g for 15 min. The supernatant was filtered through Whatman No. 1 filter paper and the filtrated (WSHM) were dried at 40◦C for about 72 h, weighed, and stored in a vacuum-dried chamber. In the WSHM, 68 aromatic, aliphatic, and nitrogen-based compounds were detected by tetramethyl ammonium hydroxide (TMAH) py-GC/MS (Gao T.G. et al., 2015).

All the bacterial strains and plasmids used in this study are listed in **Supplementary Table S1**. Among them, S. meliloti 1021 is a native mutant in which the ORF of expR is interrupted by an insertion; while S. meliloti 8530 is a derivative of S. meliloti 1021 by excising this insertion spontaneously. Since most S. meliloti strains harbor a functional expR (Pellock et al., 2002), S. meliloti 8530 was considered as the wild type strain. Agrobacterium tumefaciens KYC55 (pJZ372, pJZ384, pJZ410) was used as a bioassay strain for ultrasensitive detection of AHL.

### Growth Response of S. meliloti 8530 to WSHM

Sinorhizobium meliloti 8530 was preincubated in 50 mL of YM broth (Gao T.G. et al., 2015) for 4 days at 28◦C with shaking (150 rpm). The culture was used to inoculate 50 mL of either YM broth (control) or YM broth with 500 mg L−1 WSHM at a final OD600 of 0.01. The cultures were incubated at 28◦C with

shaking (150 rpm) for 5–6 days and samples were plated using serial dilutions every 12 h to evaluate the rhizobia cell density. This assay was performed thrice in triplicate.

### Response of AHL Production to WSHM

Agrobacterium tumefaciens KYC55 (pJZ372, pJZ384, pJZ410) was incubated in AT medium [KH2PO4, 10.7 g; MgSO4 · 7H2O, 160 mg; CaCl2, 78 mg; FeSO4 · 7H2O, 5 mg; MnSO4 · H2O, 2.2 mg; (NH4)2SO4, 2 g; glucose, 5 g; in 1 L of distilled water; pH adjusted to 7.3 with K2HPO4; tetracycline, spectinomycin, and gentamycin at final concentrations of 2, 100, and 100 µg/mL respectively] with shaking at 150 rpm for 2 days to yield up to 109 cells mL−1 (Zhu et al., 2003). The AHL production was detected as previously described (Zheng et al., 2006). Briefly, the cell-free supernatant of S. meliloti 1021 or 8530 culture (**Figure 2B**) was added to fresh AT broth at the ratio of 10% (v/v). The cell-free supernatant of A. tumefaciens strain R10 (pCF218) (Zhu et al., 2003) culture was used as positive control to assess the AHL sensitivity of A. tumefaciens KYC55 under these experimental conditions. The same volume of sterilized water (Control) or WSHM solution (500 mg L−1 ) was added separately into AT broth as negative controls. All the AT media prepared for tests were inoculated with approximately 107 cells mL−1 of A. tumefaciens KYC55 and incubated with aeration (150 rpm) for 16–20 h at 28◦C. The β-galactosidase activity in each culture was quantitatively analyzed to estimate the concentration of AHL (Zhu et al., 2003). β-galactosidase activity (Miller units) was calculated as OD420 × 103 /(time of reaction in minutes × volume of culture in milliliters × OD600) (Pérez-Montaño et al., 2011). Assays were conducted in triplicate for three times.

# Response of EPS Synthesis to WSHM Treatment

The effects of WSHM on EPS synthesis were analyzed for the wild type strain S. meliloti 8530, which can produce EPS I (succinoglycan), EPS II (galactoglucan), and linear mixed-linkage β-glucan (MLG) (Pérez-Mendoza et al., 2015), as well as for S. meliloti 1021, which could produce EPS I and extremely low level of EPS II (Pellock et al., 2002). Both strains were cultured in GMS medium (Staehelin et al., 2006) with and

without 500 mg L−1 of WSHM for 5 days. EPSs were collected as described by Staehelin et al. (2006) and quantified in triplicate by the anthrone-sulfuric acid method (Jones, 2012).

# Expression of exoY and expE Genes in Response to WSHM

The effects of WSHM on the expression of exoY and expE, which are responsible for EPS I and EPS II synthesis, respectively (Mueller and González, 2010), were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). S. meliloti 8530 was cultured in YM broth (control) or YM broth supplied with 500 mg L−1 WSHM as mentioned above. Samples were collected at the end of exponential phrase (OD600 = 1.5–2.0). Total RNA was isolated using the RNA Pure Bacteria Kit (CWBIO, Beijing, China) according to the manufacturer's instructions. First strand cDNAs were synthesized using PrimeScript Reverse Transcriptase (RT) (TaKaRa Code: D2680S) according to the manufacturer's instructions. These cDNA samples were used for RT-qPCR with primers specific to exoY and expE genes (**Supplementary Table S2**). A 167 bp fragment of 16S rRNA gene was used as an internal control for normalization (Pérez-Montaño et al., 2014). Each 20 µLreaction contained 10 µL of Power SYBR Green Master Mix (ABI, United States). The PCR program was: 95◦C for 10 min, followed by; 40 cycles of 95◦C for 15 s and 60◦C for 1 min, followed by the melting curve. PCR was performed on an ABI 7500 Thermocycler and data were analyzed using the 2−11Ct method (Jannin et al., 2012). The experiment was performed for three times with four technical replicates.

# Expression of QS Genes in Response to WSHM

The bacteria were cultured same as described above (see Expression of exoY and expE Genes in Response to WSHM). The expression of QS genes following WSHM treatment was analyzed by RT-qPCR as mentioned above with primers specific to sinI, sinR, and expR (**Supplementary Table S2**). The cDNA obtained from cultures of S. meliloti 8530, S. meliloti 1021 (1expR), S. meliloti MG170 (1sinR), and S. meliloti MG32 (1sinI) (**Supplementary Table S1**) with or without WSHM were used as templates, and the experiment was performed three times with four replicates.

### Deletion of qsrR and Its Effect on expR Expression in Response to WSHM

Agrobacterium tumefaciens interacts with plant hosts similar to those of rhizobia. Thus, in order to analyze the repression mechanisms of WSHM on expR expression, the QS regulation system in A. tumefaciens (Gonzalez and Keshavan, 2006) was compared with that in S. meliloti 8530. AccR of A. tumefaciens can counteract traR repression by directly binding with opines produced by the plant host. Since protein SMc03890 in strain 8530, which was renamed as QsrR in this study, was very similar to AccR (with 31% similarity of amino acid sequence) and were both DeoR family transcriptional regulators, we hypothesized that QsrR could be capable of mediating the effects of WSHM on expR expression. To test this hypothesis, the S. meliloti1qsrR mutant strain was constructed via homologous recombination. Furthermore, by fusing the expR promoter region with the lacZ structural gene, the change in expR expression caused by the deletion of qsrR was determined by the activity of β-galactosidase. Refer to **Supplementary Methods** for detailed description of the process.

Previous reports have evidenced that the eukaryotic hosts are capable of interfering with bacterial QS by producing molecular signals, like flavonoid (Kalia, 2013; Nievas et al., 2017), and flavonoid homologs have been detected in WSHM (Gao T.G. et al., 2015). In order to analyze whether WSHM function as a plant signal (like opines) to represses QS in S. meliloti, expR gene expression levels in S. meliloti 8530 or S. meliloti1qsrR following treatment with either WSHM (500 mg L−1 ) or alfalfa seed exudates (2%, v/v) were determined by measuring the activity of β-galactosidase (Chai et al., 2010). Alfalfa seed exudates were prepared according to Cai et al. (2009). Assays were conducted in triplicate and repeated three times.

# QsrR Purification and Electrophoretic Mobility Shift Assays (EMSAs)

The full-length ORF of qsrR was amplified using the primers qsrR281 and qsrR282 (**Supplementary Table S2**) and cloned into pET28a. The recombinant plasmid pET28a-qsrR was transformed into Escherichia coli Rosetta (DE3) and cultured in LB medium with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) for induction at 37◦C. The QsrR protein with a His tag (His6-QsrR) was purified with Ni-loaded nitrilotriacetic acid (NTA) resin (GE Healthcare) from cultures of E. coli Rosetta (DE3) carrying the recombinant plasmid. Electrophoretic mobility shift assays (EMSAs) were used to detect the interaction between the QsrR protein and the expR promoter using a DIG Gel Shift Kit, 2nd Generation (Roche), according to the manufacturer's instructions. The promoter region of expR was amplified with primers expR11 and expR12 (**Supplementary Table S2**). Binding specificity was evaluated through addition of ∼100-fold excess of unlabeled expR promoter fragments, which competed with the labeled probe to bind with His6-QsrR. A labeled non-specific DNA probe from Streptomyces avermitilis was used as negative control. EMSAs were repeated at least twice.

# Bacterial One-Hybrid Assay

The experimental procedure was similar to that of Luo et al. (2014). Briefly, the gene qsrR was amplified with primers 03890BHf and 03890BHr (**Supplementary Table S2**), excised with Not I/Bgl II, and cloned into the bait plasmid pB1H1 to generate pB1H1-qsrR. Fragments R1 and R4 in the promoter region of expR were cloned into the prey plasmid pH3U3, respectively. pH3U3-R1 and pH3U3-R4 were not self-activating prey confirmed previously (see **Supplementary Methods** for detail). Then the plasmid pairs pB1H1-qsrR/pH3U3-R1 and pB1H1-qsrR/pH3U3-R4 were transformed into E. coli USO respectively. The growth of the transformants of E. coli USO, including USO: pB1H1-zif268/pH3U3-zif268 as positive control

(+/+); USO: pB1H1/pH3U3 (−/−), USO: pB1H1/pH3U3-R1 and USO: pB1H1/pH3U3-R4 as negative controls (−/+); and two transformants with qsrR, USO: pB1H1-qsrR/pH3U3-R1 and USO: pB1H1-qsrR/pH3U3-R4 (+/+), were determined on NM medium without histidine and with varying concentrations of 3-AT. We expected the transformants with qsrR to survive on NM medium containing 3-AT, as long as QsrR interacts with the expR promoter region to recruit RNA polymerase and activate the transcription of the reporter gene HIS3. Higher levels of HIS3 gene expression enable the bacteria grow on NM medium with higher concentrations of 3-AT. Assays were repeated three times.

### Effects of WSHM on Plant Growth and Nodulation by S. meliloti

Sinorhizobium meliloti was cultured aerobically at 28◦C in YM broth (Gao T.G. et al., 2015) for 2 days to OD600 = 1.0 (about 108 CFU mL−1 ) and were used as inoculant. Medicago sativa seeds were surface-sterilized by 3% (v/v) NaClO for 3 min, germinated on 0.7% agar-water plates in the dark at 28◦C for 24–48 h. The germinated seeds were planted in pots (three seeds per pot) filled with 300 cm3 of vermiculite and moisturized with low-N nutrient solution (Vincent, 1970). Six treatments with seven replicates (i.e., pots) were included: no inoculation control with and without WSHM (500 mg L−1 ); inoculation treatments of wild type strain (S. meliloti 8530, 1 mL) with and without WSHM; and inoculation treatments of 1qsrR strain (1 mL) with and without WSHM (**Table 1**). Plants were grown in a greenhouse at 25 ± 2 ◦C during the day and 17 ± 2 ◦C at night with 60% relative humidity. Pots were rearranged daily to give a random distribution of growth conditions. After 45 days, all of the alfalfa plants were harvested and the number, fresh weight, and nitrogenase activity of the nodules, as well as the dry weight of the plants, were determined for each pot. The nitrogenase activity was analyzed by acetylene reduction assay, for which the whole roots with nodules in each pot (three plants) were put into a sealed bottle and incubated with acetylene (Suganuma et al., 1998), and the nitrogenase activity was calculated as µmol C2H4 /h · g of nodule. Then, the number and fresh weight of nodules in each pot were counted. The pooled data for each pot (three plants) were considered as one sample, and statistical analysis was conducted using Duncan test. Finally, several root nodules were sliced and treated for transmission electron microscopy (TEM) according to Bourassa et al. (2017) to evaluate the effects of WSHM treatment on the number and morphology of bacteroids in nodules induced by S. meliloti 8530.

# RESULTS

### The Growth of S. meliloti in Response to WSHM

As shown in **Figure 2A**, the cell density of S. meliloti 8530 was about 3.5 × 107 CFU/mL (83 h) in the WSHM solution or in YM-CN broth (without carbon/nitrogen source) supplied with WSHM. While it was 2.51 × 109 CFU/mL in YM broth and 3.98 × 1010 CFU/mL in YM broth supplied with WSHM, which corresponds to 14.8-fold increase in cell density by WSHM at the stationary phase (**Figure 2A**). We could calculate from these data that only 0.09% of the total cell number increased in YM broth supplied with WSHM could be attributed to the additional nutrient sources from WSHM. Therefore, this growth enhancement can be mainly attributed to the stimulation effect of WSHM.

### Synthesis of AHL in Response to WSHM

In this analysis, the AHL detector strain, A. tumefaciens KYC55 did not respond to WSHM but did respond to the AHL produced by A. tumefaciens R10 (**Supplementary Figure S1**). Thus, A. tumefaciens KYC55 was used to investigate the effects of WSHM on AHL production in S. meliloti. AHL synthesis in S. meliloti 8530 started in the beginning of exponential phase and reached a peak during the middle of exponential phase (50 h); while the AHL synthesis during the whole potential phase was significantly decreased (60–93%) by WSHM (**Figure 2B**). WSHM treatment also reduced the production of AHL in S. meliloti 1021 by 57.9% (from 65 to 28 mU/mL, 50 h of incubation) (**Supplementary Figure S1**). Additionally, increased production of AHL by A. tumefaciens R10 in response to WSHM was observed (**Supplementary Figure S1**). These results suggest that WSHM could regulate the QS system in S. meliloti.

∗Values are expressed as average ± standard deviation (n = 7). Values in the same column that have different letters are statistically significant as assessed by Duncan test (P < 0.05). #Three plants were included in each pot.

# EPS Synthesis and exoY/expE Expression of S. meliloti in Response to WSHM

fmicb-09-03194 December 21, 2018 Time: 19:3 # 6

Exopolysaccharides synthesis in S. meliloti 1021 was low (<200 µg ml−1 ) and was not affected by WSHM (**Figure 2C**); while EPS synthesis in S. meliloti 8530 was high (680 µg ml−1 ) without WSHM and was decreased by 30.87% (480 µg ml−1 ) with WSHM treatment.

For expE, a gene involved in EPS II synthesis and regulated by AHL-ExpR, its expression was down-regulated significantly by treatment with WSHM (**Figure 2D**); meanwhile, the expression of exoY, a gene involved in EPS I synthesis, was not affected by WSHM in S. meliloti 8530. All of these results suggest that WSHM did not affect the synthesis of EPS I, but decreased the production of EPS II and MLG.

# Impact of WSHM on the Expression of Genes Involved in QS

In this analysis, WSHM treatment significantly down-regulated the expression of sinI, sinR, and expR in S. meliloti 8530 (**Figure 3A**); while in the expR mutant strain, S. meliloti 1021, WSHM only down-regulated sinR expression and did not alternate sinI expression (**Figure 3B**). In addition, the expression levels of both sinI and expR were down-regulated by WSHM in the sinR mutant (MG170, **Figure 3C**). These results suggest that the repression of sinI in S. meliloti 8530 by WSHM is due to the repression of expR and is independent of sinR. In the sinI mutant (MG32), the expression levels of the sinR and expR genes were down-regulated by WSHM (**Figure 3D**); suggesting that the effects of WSHM on sinR and expR expression are independent of sinI. Furthermore, the sinR expression was down-regulated by WSHM in both S. meliloti 8530 and S. meliloti 1021 (1expR), suggesting that the repression of sinR expression by WSHM is independent of ExpR.

# Effects of WSHM on the Expression of expR in S. meliloti 1qsrR

The expression level of the expR gene in S. meliloti1qsrR (SMc03890 deletion, see Section "Deletion of qsrR and Its Effect on expR Expression in Response to WSHM" for detail) was significantly higher than that in the WT (**Figure 4**), suggesting that QsrR represses expR transcription. Thus, the SMc03890 gene was renamed qsrR, for "quorum sensing regulator expR repressor." As expected, the expR gene was down-regulated

significantly in response to WSHM in S. meliloti 8530, but not in S. meliloti1qsrR, suggesting that QsrR is responsible for repressing expR expression in response to WSHM. Meanwhile, alfalfa seed exudates significantly repressed expR expression in both S. meliloti 8530 and S. meliloti1qsrR (**Figure 4**), suggesting that the repression of expR expression by seed exudates is independent of QsrR.

# Potentiated Interaction Between QsrR and expR Promoter by WSHM

In order to test whether QsrR regulate the expression of expR directly or indirectly, EMSA were performed. As shown in **Figure 5**, a retarded DNA was observed when His6-QsrR (3.3 µM) was added to the assay mixture, indicating that QsrR was able to bind directly with the promoter region of expR gene. There was no retardation in the binding specificity assay or the negative control. These findings indicate that the transcription of expR is directly regulated by QsrR. In the EMSA test that determined the effect of WSHM on the interaction between QsrR and expR promoter, no band was detected when WSHM was added into the reaction system (data not shown). This might result from the possibility that WSHM interfered with the experimental process of EMSA, such as electrophoresis or binding of DNA with nylon membrane. Therefore, the role of WSHM in regulating expR expression via QsrR was further investigated by a bacterial one-hybrid assay (see **Supplementary Methods** and **Supplementary Figure S3** for detail). The negative control strains E. coli USO (pB1H1/pH3U3-R1) and USO (pB1H1/pH3U3-R4) did not express qsrR and grew poorly; meanwhile, the positive strains USO (pB1H1-qsrR/pH3U3-R1) and USO (pB1H1-qsrR/pH3U3-R4) expressed qsrR and grew much better than the negative controls on selective medium (NM+3-AT) (**Figure 6C**). The positive strains were able to grow on medium with 2 mM of 3-AT that confirmed the ability of QsrR to bind with the promoter region of the expR gene. The binding region of QsrR is closer to the start of SMc03900 gene rather than that of expR (region R4, **Figure 6B**). In addition, the growth of the positive strains was significantly enhanced when WSHM were added to the selective medium; tolerance to 3-AT also increased from 2 to 5 mM. Meanwhile, growth of the negative control strains (USO: pB1H1/pH3U3-R1 and USO: pB1H1/pH3U3-R4) was not enhanced by WSHM on NM medium containing either 3 or 5 mM of 3-AT, further confirming that WSHM do not serve as a source of nutrients for these strains. These results suggest that QsrR can bind with the promoter region of expR gene, while WSHM can significantly potentiate this interaction.

### Effects of WSHM on M. sativa Growth and Nodulation With S. meliloti 8530

Water-soluble humic materials treatment increased plant dry weight by 5.4% compared to the control (**Table 1**). The nitrogenase activity and plant dry weight of M. sativa inoculated with S. meliloti 8530 and 500 mg L−1 of WSHM were significantly increased by 56.4 and 90.36%, respectively, compared with plants inoculated only with S. meliloti 8530, while no statistically significant difference was detected in nodule number and nodule fresh weight (13.1 and 29.9%) between these two treatments. No significant difference was observed in nitrogenase activity, plant dry weight, nodule number, or nodule fresh weight between the M. sativa inoculated with S. meliloti1qsrR +/− WSHM. Besides, the nitrogenase activity of plants inoculated with S. meliloti1qsrR alone was significantly increased by 69.53% compared with plants inoculated with S. meliloti 8530.

Transmission electron microscopy was performed to tested whether the increased nitrogenase activity of M. sativa inoculated with S. meliloti 8530 by WSHM was due to increased bacteroid density in nodules. The TEM images revealed that the number and morphology of bacteroids in alfalfa nodules induced by S. meliloti 8530 were not impacted by WSHM treatment (**Supplementary Figure S4**). Thus, WSHM may have affected the nitrogenase activity of the root nodules by other mechanisms, such as regulating the expression of nitrogen fixation genes or improving the energy supply to the bacteroids.

# DISCUSSION

Quorum sensing regulates metabolically costly cooperative behaviors of bacteria depending on the environmental and physiological characteristics, such as production of exopolysaccharides, motility, and life-style switches related to symbiosis with eukaryotic hosts (Hense and Schuster, 2015; Calatrava-Morales et al., 2018). Many plant-associated bacteria communicate with the plant host through QS (González and Venturi, 2013; Schikora et al., 2016). The ExpR/Sin system is the sole QS system in S. meliloti 8530 (Krol and Becker, 2014) (**Figure 1**) that is involved in the regulation of metabolic and symbiotic procedures (Hoang et al., 2004; Gurich and Gonzalez, 2009). Our study regarding the effects of WSHM on the ExpR/Sin system in S. meliloti 8530 yielded several important findings.

SMc03890, renamed as qsrR, was shown to code a protein that directly represses expR gene transcription (**Figures 4–6**). As a versatile LuxR homolog regulator, ExpR directly or indirectly regulates the expression of at least 570 genes and plays a central role in the QS network in S. meliloti (Hoang et al., 2004; Gurich and Gonzalez, 2009; Charoenpanich et al., 2013). Thus, qsrR might have an essential function in different processes of S. meliloti. Previously, few studies on the regulation of the expR gene have been performed. One previous study showed that L-canavanine can repress expR expression as an arginine analog (Keshavan et al., 2005); meanwhile, Gao M. et al. (2015) reported that the RNA-binding protein Hfq regulates expR post-transcriptionally at higher population densities. Our study described QsrR as a novel repressor for expR transcription (**Figure 4**) and revealed the physical interaction between QsrR and the expR promoter by EMSA (**Figure 5**) and one-hybrid assays (**Figure 6C**). In addition, the increased nitrogenase activity in the nodules formed by S. meliloti1qsrR compared with those formed by S. meliloti 8530 (**Table 1**) suggests that QsrR may also regulate the expression of other genes, including those involved in nitrogen fixation. The function of QsrR is worthy of further research due to the versatile role of ExpR in regulating the metabolism and nodulation of S. meliloti. For example, the specificity of DNA sequences that can bind with QsrR might be further studied in order to reveal details in the interaction between QsrR and expR promoter.

The regulation of QS system in S. meliloti by WSHM via repressing expR expression (**Figures 2–4**) was evidenced for the first time, although different biological effects of humic materials on plants (Jannin et al., 2012; Traversa et al., 2013; Canellas et al., 2015; Savy et al., 2017) and bacteria (Tikhonov et al., 2010) have been demonstrated. The results from **Figures 4–6** demonstrated that QsrR may mediate the repression of expR expression by WSHM. It is possible that WSHM also repress sinR expression via additional mechanisms since QsrR cannot bind with the promoter region of sinR (**Supplementary Figure S2**). Moreover, our results suggest that WSHM function in a manner comparable to alfalfa seed exudates and may act as plant signal to repress expR gene expression in S. meliloti (**Figure 4**). Previous reports have demonstrated that plants are able to produce compounds that mimic or inhibit bacterial QS processes to promote their development of beneficial traits (Gao et al., 2003; Schikora et al., 2016; Nievas et al., 2017). WSHM might help plants to regulate bacterial QS and improve the symbiotic relationship between the host plant and bacteria. Compounds such as furan, pyrrole, benzpyrole, benzene rings, and esters have been identified in WSHM (Gao T.G. et al., 2015) and they may interfere with microbial QS systems (Gonzalez and Keshavan, 2006; Christensen et al., 2013). However, further investigation is needed to identify the compounds responsible for S. meliloti QS regulation in WSHM or alfalfa seed exudates.

In addition, the results suggest that the inactivation of rhizobia ExpR/Sin genes in nodules might be due to regulation by the plant host. Even though the QS system in S. meliloti has been reported to control cell functions essential for successful plant invasion, the ExpR/Sin genes were inactive in nodules due to an unknown mechanism (Gurich and Gonzalez, 2009). Alfalfa seed exudates and WSHM repressed the expression of expR in S. meliloti (**Figure 4**), suggesting that the inactivity of ExpR/Sin genes in nodules is a response of rhizobia to the host signal molecules. The repression of expR by host signal might due to the presence of L-canavanine in alfalfa seed exudates and L-canavanine could

cause misfolding of the ExpR protein (Keshavan et al., 2005), while ExpR has been reported to possess the potential for selfregulation (Charoenpanich et al., 2013).

Water-soluble humic materials can stimulate S. meliloti growth by repressing QS. Although WSHM contain 52.18% C and 3.72% N (Gao et al., 2012) and humic acids could act as C or N source for bacteria growth (Tikhonov et al., 2010; Gao T.G. et al., 2015), only 0.09% of the increased biomass in WSHM treatment could be contributed to the C/N supply by WSHM in YM broth (**Figure 2A**). Thus, we conclude that WSHM increase S. meliloti 8530 growth mainly due to the regulatory effects on cell metabolism. The repression of the QS system in S. meliloti 8530 by WSHM could stimulate growth (**Figures 2**, **3**), which supported the observation that QS restrains growth in S. meliloti, M. huakuii, R. leguminosarum, and Rhizobium sp. NGR234 (Wilkinson et al., 2002; He et al., 2003; Gao et al., 2006; Charoenpanich et al., 2015).

Water-soluble humic materials could regulate EPS synthesis, mainly decrease EPS II and MLG production, but do not affect EPS I production (**Figures 2C,D**). Amongst the three kinds of EPS produced by S. meliloti 8530 (Pérez-Mendoza et al., 2015), EPS I is the most efficient compound at initiating and maintaining infection threads (Jones, 2012). The probability of causing aberrant infection threads by EPS II, which is less

efficient than EPS I at inducing infection thread formation, is 8–10 times higher than that of EPS I (Pellock et al., 2000). Furthermore, EPS synthesis in rhizobia is energy consuming; thus, EPS II is not required if EPS I is present (Zhan et al., 1989; Mithöfer, 2002). Even though MLG is involved in the attachment of S. meliloti to alfalfa roots, there was no significant difference in nodulation occupancy between a wild-type and a MLG synthesis deficient mutant (Pérez-Mendoza et al., 2015). Therefore, EPS II and MLG are not likely to be essential for efficient nodulation between S. meliloti and alfalfa. This notion is supported by the fact that the nodule occupancy of S. meliloti 1021 (1expR) was 10–20% higher than that of S. meliloti 8530 (Charoenpanich et al., 2015). Thus, shutting down EPS II and MLG production with WSHM may promote symbiotic nitrogen fixation between S. meliloti and M. sativa. It is noteworthy that the ExpR/Sin QS system can regulate EPS II and MLG production (Mueller and González, 2010; Pérez-Mendoza et al., 2015), while EPS I biosynthesis becomes independent of QS in the absence of ExpR, and mutations to expR did not alter the amounts of EPS I produced (Glenn et al., 2007). This may be because MucR increases the production of EPS I independently of QS (Mueller and González, 2010). Thus, WSHM may regulate EPS production in S. meliloti 8530 through their regulation of the QS system.

Water-soluble humic materials improved symbiotic nitrogen fixation between M. sativa and S. meliloti (**Table 1**). Since WSHM did not affect the density of bacteroids in nodules (**Supplementary Figure S4**), the increase in nitrogenase activity of M. sativa following treatment with WSHM (**Table 1**) may be due to the enhanced expression of nifA gene via repressing expR expression. It has been reported that AHL-ExpR repress the expression of genes related to nitrogen fixation, such as fixTQK, which induce the expression of nifA (Hoang et al., 2004; Charoenpanich et al., 2013). In addition, the failed of significant increase in nodule number and nodule fresh weight of M. sativa by WSHM may result from the fact that nodule number and nodule weight are controlled by the plant host to ensure optimal growth (Mortier et al., 2012; Zipfel and Oldroyd, 2017). The promotion of nitrogen fixation between S. meliloti and M. sativa by WSHM offers an economical and efficient route for improving biological nitrogen fixation in agriculture. In a field experiment, WSHM treatment (500 mg/mL) on seeds (375 g WSHM per ha) increased alfalfa yield by 29% per year (unpublished data).

### CONCLUSION

We identified QsrR as a direct repressor of expR (a gene central to the QS system in S. meliloti 8530) transcription. WSHM

# REFERENCES

were proposed to repress expR expression by modulating the interaction between QsrR and the expR promoter, and ultimately decrease AHL, EPS II and MLG production. Consequently, WSHM increased the growth of S. meliloti, as well as symbiotic nitrogen fixation with M. sativa. In addition, both expR and sinR were down-regulated by WSHM with independent mechanisms. We hypothesize that QsrR may mediate the repression of expR expression by WSHM; however, the mechanism by which WSHM down regulate sinR expression requires further investigation.

# AUTHOR CONTRIBUTIONS

Y-YX, J-SY, CL, and H-LY conceived and designed the study. Y-YX, R-NW, and X-QQ performed the experiments. Y-YX and E-TW wrote the manuscript. Y-YX, and B-ZL participated in the preparation of water-soluble humic materials. H-LY and W-FC helped to design the experiments and drafted the manuscript.

# FUNDING

This work was supported by the National Natural Science Foundation of China (Grant No. 31770541), the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201403048-2) and by the fund for Shanxi "1331 Project" Collaborative Innovation Center.

# ACKNOWLEDGMENTS

We thank Mengsheng Gao for providing S. meliloti1sinI and 1sinR mutant strains, Li Luo for providing strain S. meliloti 8530, Scot A. Wolfe and Zhi Chen for providing bacterialone-hybrid system, and Jun Zhu for providing Agrobacterium tumefaciens KYC55 (pJZ372, pJZ384, and pJZ410) and R10 (pCF218). We would also like to thank Lijie Sun for the very helpful discussions.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03194/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Xu, Yang, Liu, Wang, Wang, Qiu, Li, Chen and Yuan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# SNARE Proteins LjVAMP72a and LjVAMP72b Are Required for Root Symbiosis and Root Hair Formation in Lotus japonicus

Aoi Sogawa1† , Akihiro Yamazaki2† , Hiroki Yamasaki1 , Misa Komi1 , Tomomi Manabe1 , Shigeyuki Tajima1 , Makoto Hayashi2 and Mika Nomura1 \*

1 Faculty of Agriculture, Kagawa University, Kagawa, Japan, 2 RIKEN Center for Sustainable Resource Science, Yokohama, Japan

SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins mediate membrane trafficking in eukaryotic cells. Both LjVAMP72a and LjVAMP72b are members of R-SNARE and belong to a symbiotic subgroup of VAMP72 in Lotus japonicus. Their sequences are closely related and both were induced in the root upon rhizobial inoculation. The expression level of LjVAMP72a in the nodules was higher than in the leaves or roots; however, LjVMAP72b was expressed constitutively in the leaves, roots, and nodules. Immunoblot analysis showed that not only LjVAMP72a but also LjVAMP72b were accumulated in a symbiosome-enriched fraction, suggesting its localization in the symbiosome membrane during nodulation. Since there was 89% similarity between LjVAMP72a and LjVAMP72b, knockdown mutant by RNAi suppressed both genes. The suppression of both genes impaired root nodule symbiosis (RNS). The number of bacteroids and the nitrogen fixation activity were severely curtailed in the nodules formed on knockdown roots (RNAi-LjVAMP72a/72b). Arbuscular mycorrhization (AM) was also attenuated in knockdown roots, indicating that LjVAMP72a and LjVAMP72b were required to establish not only RNS but also AM. In addition, transgenic hairy roots of RNAi-LjVAMP72a/72b suppressed the elongation of root hairs without infections by rhizobia or arbuscular mycorrhizal fungi. Amino acid alignment showed the symbiotic subclade of VAMP72s containing LjVAMP72a and LjVAMP72b were a conserved six amino acid region (HHQAQD) within the SNARE motif. Taken together, our data suggested that LjVAMP72a and LjVAMP72b positively controlled both symbioses and root hair formation by affecting the secretory pathway.

Keywords: Lotus japonicus, nitrogen fixation, root hair, SNARE, symbiosis, VAMP72

# INTRODUCTION

Legume-rhizobia interaction has developed from complex signal exchange. Upon sensing host plant-derived flavonoids, rhizobia produce and secrete lipochito-oligosaccharides called nodulation factors (nod factors), which are then recognized by the host and trigger multiple events leading to establishing root nodule symbiosis (RNS) (Dénarié et al., 1996; Oldroyd, 2013). During nodulation, bacterial infection and nodule organogenesis take place in epidermal and cortical cells, respectively (Suzaki and Kawaguchi, 2014). Nod factors trigger deformation and curling of root hairs. Rhizobia then penetrate into root hairs via infection threads (Kouchi et al., 2010;

### Edited by:

Kiwamu Minamisawa, Tohoku University, Japan

### Reviewed by:

Norio Suganuma, Aichi University of Education, Japan Fan Chen, Institute of Genetics and Developmental Biology (CAS), China

\*Correspondence:

Mika Nomura<EMAIL_ADDRESS>†These authors have contributed equally to this work

### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

Received: 13 September 2018 Accepted: 20 December 2018 Published: 16 January 2019

### Citation:

Sogawa A, Yamazaki A, Yamasaki H, Komi M, Manabe T, Tajima S, Hayashi M and Nomura M (2019) SNARE Proteins LjVAMP72a and LjVAMP72b Are Required for Root Symbiosis and Root Hair Formation in Lotus japonicus. Front. Plant Sci. 9:1992. doi: 10.3389/fpls.2018.01992

**66**

Oldroyd, 2013). Simultaneously, cell division occurs in the cortical layer to form nodule primordia (Oldroyd, 2013). When infection threads reach the nodule primordia, rhizobia are released from the infection threads to nodule primordia by an endocytosis-like process, in which rhizobial cells are surrounded by host-derived membranes [symbiosome membrane (SM)] called a "symbiosome" (Newcomb, 1976; Verma et al., 1978). In addition to RNS, legumes establish an endosymbiotic association with arbuscular mycorrhizal fungi (AMF). AMF secrete sulfated and non-sulfated lipochito-oligosaccharide signals (Myc-LCOs), which function as signals for allowing AMF to enter host roots (Maillet et al., 2011). AMF penetrate the root epidermis by forming hyphopodia on the root surface and extend their internal hyphae toward the inner cortex, where they form arbuscules that are surrounded by a plant plasma membrane called a periarbuscular membrane (PAM). These dynamic changes to form a SM and PAM are closely associated by membrane trafficking of endoplasmic reticulum (ER) and/or Golgi apparatus and their transport vesicles (Robertson et al., 1978; Newcomb and McIntyre, 1981; Roth and Stacy, 1989; Genre et al., 2012).

SNARE (soluble NSF attachment protein receptor) proteins are known to mediate the transport vesicle, and its molecules have a highly conserved coiled-coil domain known as a SNARE motif (Harbury, 1998; Jahn et al., 2003). The SNARE-mediated membrane trafficking forms SNARE complexes with R-SNARE on the vesicle and two or three Q-SNAREs on the target membrane. During the past decade, it has been clear that SNARE proteins play roles in general homeostatic and housekeeping functions within the cell. In Arabidopsis, two plasma membranelocalized Qa-SNAREs, SYP123 and SYP132, mediate membrane trafficking for root hair formation (Ichikawa et al., 2014). Recently, a symbiotic Qa-SNARE protein, SYP132A, has been shown to be generated by alternative termination of transcription and is essential for the formation of both SM and PAM in Medicago truncatula (Huisman et al., 2016; Pan et al., 2016). R-SNAREs located on the transport vesicles are classified into three groups: Sec22, YKT6, and VAMP7 (McNew et al., 1997; Galli et al., 1998; Gonzalez et al., 2001). VAMP7 consists of two major subgroups in plants: VAMP71 and VAMP72 (Sanderfoot, 2007). VAMP71 is known to have a similar function to mammalian VAMP7s; however, another group, VAMP72, appears to be specific to green plants (Sanderfoot, 2007). The Arabidopsis VAMP72 proteins except AtVAMP727 are localized in the plasma membrane (Marmagne et al., 2004; Uemura et al., 2004). To determine the specific function for symbiosis, it is better to focus on VAMP72 in legume plants. In M. truncatula, VAMP721d and VAMP721e are required for both RNS and arbuscular mycorrhization (AM) (Ivanov et al., 2012). For Lotus japonicus, there are reports on SNARE-mediated membrane trafficking in nodules. The sed5-like LjSYP132-1, Qa-SNARE, contributes to nodule formation and plant growth development (Mai et al., 2006). SYP71, Qc-SNARE, plays a role in symbiotic nitrogen fixation (Hakoyama et al., 2012). However, there is no report on R-SNARE. In this study, we focused on an R-SNARE, VAMP72, in the formation of symbiotic interfaces. Our data revealed that LjVAMP72a and LjVAMP72b were required for both RNS and AM as well as for root hair development.

# MATERIALS AND METHODS

### Plant and Microbial Materials

Seeds of L. japonicus B129 Gifu (Handberg and Stougaard, 1992) were surface sterilized with 3% sodium hypochlorite and then germinated. Six-day-old seedlings were transplanted onto sterile vermiculite with a half concentration of B&D medium (Broughton and Dilworth, 1971), followed by inoculation with Mesorhizobium loti strain MAFF303099 at 7 days after transplanting. Plants were grown in a growth chamber at 24◦C under a 16 h light/b h dark condition.

# RNA Extraction and Real-Time PCR Analysis

Total RNA was isolated from various organs of L. japonicus using the RNeasy Plant Mini Kit according to the manufacturer's instructions (Qiagen, Tokyo, Japan). The cDNA was synthesized using PrimeScript RT Master Mix (Takara, Shiga, Japan). Quantitative RT-PCR reactions were performed in triplicate on cDNA (equivalent to about 100 ng of RNA) using the SYPR premix Ex Taq (Takara), and real time detection was performed on a Thermal Cycler Dice Real Time System II (Takara) with the primer set shown in **Supplementary Table S1**. Ubiquitin was used as an internal standard. All data were normalized because the expression levels of ubiquitin are equal in each sample. All experiments were performed with more than three biological replications.

# Orthology Prediction and Phylogeny Reconstruction

Putative orthologs of MtVAMP72d were searched for in a database consisting of genomes and transcriptomes (i.e., all available CDSs) of 70 green plant species (Sato et al., 2008; Carleton College, and The Science Education Resource Center, 2010; Goodstein et al., 2012; Dash et al., 2016) (see **Supplementary Table S1** for the complete list). OrthoReD (Battenberg et al., 2017) was used to predict the orthologs of MtVAMP72d with its amino acid sequence as a query against the database without setting any outgroups. Based on the phylogenetic tree reconstructed by OrthoReD, a clade of putative orthologs of MtVAMP72d in several species (A. thaliana, Glycine max, L. japonicus, M. truncatula, Solanum lycopersicum, Populus trichocarpa, and Vitis vinifera following Ivanov et al., 2012) was determined manually. Gene names corresponding to Gene ID used in this study were listed in **Supplementary Table S2**.

The phylogeny of these putative orthologs was reconstructed. Amino acid sequences of these putative orthologs were aligned using MAFFT v7.407 (Katoh and Standley, 2013). Based on the multiple sequence alignment, the most likely phylogeny was reconstructed using RAxML v8.2.12 (Stamatakis, 2006). The best of four parallel runs was chosen as the most likely tree to avoid having the tree lodged onto a local optimum. Rapid bootstrapping of 100 replicates was also carried out using RAxML v8.2.12.

### DNA Sequencing

fpls-09-01992 January 16, 2019 Time: 13:36 # 3

Isolated cDNA and PCR amplified products were sequenced according to the manufacturer's instructions for the DNA sequencer (Model 3700; Applied Biosystems, CA, United States). The CDS of LjVAMP72b was speculated by genome sequence (Sato et al., 2008) and nodule cDNA was amplified with following primers; FW, 50 -ATGGGTCAGAACCAGAGATC-3 0 ; RV, 50 -CCTTTCCTCGCATAATCACAA-30 using Tks Gflex DNA polymerase (Takara, Japan). The resulting PCR product was sequenced from both strands.

# Construction of LjVAMP72a prom::GUS, RNAi-LjVAMP72a/72b, and LjVAMP72a-GFP

To construct the LjVAMP72a promoter fused to the GUS gene, the GUS-NOS gene was first cloned into the XbaI/SalI sites of pC1300GFP, kindly provided by Dr. H. Kouchi (National Institute of Agricultural Sciences, Tsukuba), producing the GUS-NOS in pC1300 GFP. The 50 -flanking regions [−1,817 to before ATG of the translation-initiation site in the LjVAMP72a gene (genome clone; CM0066)] was then cloned into the GUS-NOS in pC1300GFP, following the production of LjVAMP72a prom::GUS.

To construct RNAi-LjVAMP72a/72b, the cDNA of LjVAMP72a was amplified using PCR from an EST clone MR100a03 (accession No. BP083622) with the following primers: RNAi-f, 50 -ATG GAT CCC TCG AGA ATG GCT ACA CAT ATT-30 and RNAi-r, 50 -ATA TCG ATG GTA CCT TTT GGG AAT CCA CGA-30 . The amplified cDNA fragments for the RNAi construct was subcloned into the appropriate cloning sites of pC1300GFP. The procedure for constructing it has been described previously (Shimomura et al., 2006). Since the amplified cDNA has high similarity between LjVAMP72a and LjVAMP72b, the knockdown mutant suppressed both genes. Therefore the mutant was named as RNAi-LjVAMP72a/72b.

To construct LjVAMP72a-GFP, used by Arabidopsis suspension cells, LjVAMP72a cDNA was fused to the sGFP under the control of CaMV35S-sGFP (S65T)-NOS30 (Niwa et al., 1999), provided by Dr. Y. Niwa, University of Shizuoka.

### Hairy Root Transformation

Hairy root transformation of L. japonicus Gifu using Agrobacterium rhizogenes LBA1334 was performed according to a procedure described previously (Kumagai and Kouchi, 2003). Transgenic hairy roots with GFP fluorescence were selected and transferred to vermiculite pots with a half concentration of B&D medium. Plants were grown in a growth cabinet at 24◦C (16 h light/b h dark). After 1 week, the plants were transferred to pots and then inoculated with M. loti MAFF303099 and continued to grow in the same medium.

### Histochemical GUS Staining

Hairy roots transformed with the LjVAMP72a prom::GUS construct were stained for 2 h at 37◦C as described previously (Mai et al., 2006). GUS-stained nodules were observed using a DM6 upright microscope (Leica, Germany) with a differential interference contrast mode. Mycorrhized roots were embedded in 5% agarose, sliced in b0 µm sections using a Zero 1 super microslicer (D.S.K., Japan), and observed using the DM6 (Leica).

### Transient Expression of the GFP-Fused Protein, LjVAMP72a-GFP, in Arabidopsis Suspension Cells

LjVAMP72a cDNA was fused to the sGFP of a CaMV35SsGFP (S65T)-NOS30 vector, producing the LjVAMP72a-GFP. RFP-tagged Arabidopsis AtSYP722, AtSYP722-RFP, was kindly provided by Dr. T. Ueda, University of Tokyo. The transient expression of LjVAMP72a-GFP and AtSYP722-RFP in Arabidopsis suspension culture cells was performed following a previous report (Ueda et al., 2001).

### Detection of LjVAMP72a and LjVAMP72b Proteins

Plant tissue (0.1 g) was harvested after 35 days infection and homogenized with a mortar and pestle in extraction buffer containing 250 mM Tris-HCl (pH 7.5), 700 mM sucrose,

100 mM KCl, 2% (w/v) insoluble polyvinylpolypyrrolidone, 2% 2-mercaptoethanol, and 2% SDS. After total maceration, water-saturated phenol was added and then centrifuged at 3,000 × g for 20 min. The phenol fraction was isolated and five volumes of methanol plus 100 mM ammonium acetate including 10 mM 2-mercaptoethanol were added. After incubation at −80◦C for 2 h, the solution was centrifuged at 15,000 × g for 5 min, and the pellets were resuspended with sample buffer [50 mM Tris-HCl (pH 6b), 100 mM DTT, 2% SDS, and 10% glycerol]. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the procedure of Nomura et al. (2006). Immunoblot analysis was performed with rabbit antisera raised against a protein of (ENIEKVLDRGEKIE) antibody as the primary antibody and detected using ECL and a western blotting detection system (GE Healthcare, United Kingdom). Since the produced antibody can detect both proteins, the

resulting antibody was named anti-LjVAMP72a/72b antibody. The protein concentration was determined using the Lowry method (Lowry et al., 1951). Lb antibody raised against rabbit was kindly provided by Prof. N. Suganuma, Aichi University of Education, Japan.

# Isolation of Symbiosome Fraction From the Nodules

Fresh nodules (10–15 g) at 4 weeks post infection were used to isolate the symbiosome fraction. The symbiosome fraction was isolated from Percoll discontinuous gradients (Day et al., 1989).

using anti-LjVAMP72a/72b antibody (F) or anti-Lb antibody (G). Lane 1: crude extract from nodules, lane 2: supernatant of the crude extract, lane 3: symbiosome-enriched fraction of a Percoll-mediated equilibrium density-gradient centrifugation. Arrowheads indicate LjVAMP72a/72b (F) and Lb (G), respectively.

In brief, nodule homogenate was centrifuged at 1,000 rpm for 15 min, and the pellet was then suspended with washing medium and further centrifuged at 2,000 rpm for 10 min. The resulting pellet was used for Percoll discontinuous gradients (80, 60, and 45%), and loose pellets were collected from the bottom of the 80% layer and the 60/80% interface as a symbiosome fraction. The isolated symbiosome fraction was suspended in washing medium and used for immunoblot analysis. After centrifugation at 1,000 and 2,000 rpm, both supernatants were used as a soluble fraction.

### Assay for Acetylene Reduction

For acetylene reduction activity, transgenic hairy root nodules were selected under fluorescence microscopy because the GFP gene was inserted in the construct vectors. The activity of GFP-positive nodules was measured using gas chromatography (Shimadzu GC-8A; Kyoto, Japan) as previously described (Suganuma et al., 1998). Experiments were performed with more than three biological replications.

# Root Hair Phenotype in RNAi-LjVAMP72a/72b Knockdown Roots

Hairy roots of L. japonicus expressing the LjVAMP72a and LjVAMP72b knockdown construct (RNAi-LjVAMP72a/72b) were observed using the DM6 (Leica). Pictures were tiled using Microsoft Image Composite Editor (Microsoft, United States). The length of the root hairs was measured from a position 1 mm from the root tip. The length of 50 root hairs per transgenic plant was measured using Image J. Experiments were performed with more than three biological replications.

# Mycorrhization Assay

Lotus japonicus expressing the RNAi-LjVAMP72a/72b construct or GFP only (empty vector) in the hairy roots were planted in plastic pots filled with sterile vermiculites in 1/2 Hoagland culture medium and inoculated with Rhizophagus irregularis (Premier Tech, Rivière-du-Loup, Canada) at 100 spores/plant. At 6 weeks after inoculation, the roots were stained with 0.05% trypan blue, and AM events were numbered and calculated using the line intersect method (McGonigle et al., 1990). Pictures of AMFinoculated roots were taken using the DM6 (Leica) after staining.

Analysis of Nodulation and Bacteroid Number in Transgenic Hairy Roots

Different colors indicate independent transgenic hairy roots.

Hairy roots were inoculated with M. loti strain MAFF303099 expressing a red fluorescent protein from Discosoma sp. (DsRed) (Maekawa et al., 2009) and used in our nodulation test. Transgenic hairy roots were selected by GFP fluorescence and observed under a fluorescence microscope (Olympus SZX9). After infection of DsRed-labeled M. loti, bacteroid was isolated from the nodule and the number of bacteroid in each transgenic nodule was counted using fluorescent microscope BX51 (Olympus).

# RESULTS

### LjVAMP72a and LjVAMP72b Were Induced Upon Rhizobial Inoculation

The genome database of L. japonicus1 possesses several VAMP72 proteins from LjVAMP72a to LjVAMP72f, LjVAMP724 and LjVAMP727. We found that one of the LjVAMP72s, LjVAMP72a in L. japonicus, was expressed in the nodules, induced upon rhizobial inoculation, and peaked at 14 days after inoculation (**Figure 1**). In addition, the genome database contains another LjVAMP72, LjVAMP72b, which has high homology to LjVAMP72a. There was 89% similarity between LjVAMP72a and LjVAMP72b. The expression of the LjVAMP72a genes was induced in the infected roots and nodules; however, LjVAMP72b was expressed constitutively in the leaves, roots, and nodules (**Figure 1**).

The phylogenic analysis (**Figure 2**) showed that both LjVAMP72a and LjVAMP72b belong to the "symbiotic" subgroup and were orthologous to MtVAMP721d and MtVAMP721e, respectively, which have been reported to function in symbioses (Ivanov et al., 2012).

The GUS expression by the LjVAMP72a promoter was detected at the vascular bundle in non-infected roots (**Figure 3A**). The expression was strongly induced in nodule primordia upon rhizobial inoculation besides its basal expression at the vascular bundles of the roots (**Figure 3B**). The GUS induction at the vascular bundle of infected roots was detected with longer staining (data not shown). The higher GUS expression by the LjVAMP72a promoter in the nodules than that in the roots was identical to that expression level of the LjVAMP72a in nodules was higher than that in roots at 28 days after infection (**Figure 1**). AM also positively affected LjVAMP72a promoter activity. During AM, LjVAMP72a was induced in cells where arbuscules formed (**Figure 3C**). These data suggested that the LjVAMP72a plays a role in both symbioses, RNS and AM. Because the genome sequence of LjVAMP72b was not completed, we have not constructed the LjVAMP72b promoter fused to the GUS gene.

# Localization of LjVAMP72a and LjVAMP72b in RNS

Uemura et al. (2004) identified AtVAMP722 SNARE localized in the plasma membrane in Arabidopsis suspension cells. **Figure 4** shows LjVAMP72a co-localization with AtVAMP722 in

1http://www.kazusa.or.jp/lotus/

suspension culture cells of A. thaliana, indicating its localization in plasma membrane (**Figures 4A–D**). We have produced a peptide antibody that can detect both LjVAMP72a and LjVAMP72b. After isolating the symbiosome-enriched fraction, immunoblot analysis was performed (**Figure 4**). Since the deduced molecular weight of LjVAMP72a and LjVAMP72b was similar (ca. 25 kDa), one band was detected at the deduced molecular size in the symbiosome-enriched fraction (**Figure 4F**, Lane 3). The LjVAMP72a and LjVAMP72b were not fractionated in the soluble fractions in which leghaemoglobin was detected (**Figure 4G**, Lanes 1 and 2). SM contains some of components present in the plasma membrane and the results showing its localization in the plasma membrane were obtained in Arabidopsis suspension cells. Taken together, these data indicated that LjVAMP72a and LjVAMP72b localized in the SM.

### LjVAMP72a and LjVAMP72b Functioned in Symbioses

We made the transgenic hairy roots of knockdown LjVAMP72a genes. RNA interference of LjVAMP72a was confirmed using qRT-PCR (**Figure 5A**). Immunoblot analysis showed that no band could be detected in a knockdown mutant, suggesting that both LjVAMP72a and LjVAMP72b were suppressed in the knockdown mutant (**Figure 5B**). Therefore, the knockdown mutant was named as RNAi-LjVAMP72a/72b.

The number of mature nodules was significantly lower in the RNAi-LjVAMP72a/72b roots compared with that in the roots transformed with an empty vector (**Figures 5C,G–L**). On the other hand, the number of developing nodules of RNAi-LjVAMP72a/72b increased, and the number of bacteroids and acetylene reduction activity were severely attenuated in the RNAi-LjVAMP72a/72b roots (**Figures 5D,E**).

LjVAMP72a and LjVAMP72b also positively affected AM (**Figures 5F,M–O**). Frequencies of the internal hyphae invasion, arbuscular and vesicle formation were significantly impaired in the RNAi-LjVAMP72a/72b knockdown roots (**Figure 5F**). **Figure 5N** shows the external hyphae did not invade the roots. Some of the infected roots only exhibited internal hyphae with almost no arbuscule when LjVAMP72a/72b was knocked-down (**Figure 5O**), whereas the empty vector control barely affected arbuscule formation (**Figure 5M**). These data speculate the symbiotic membranes would be developed by the vesicle trafficking through the LjVAMP72a and LjVAMP72b, resulting that the RNAi-LjVAMP72a/72b knockdown attenuated symbioses. These results were in line

with a previous report in which the VAMP721d and VAMP721e double knockdown attenuated the number of nodules in M. truncatula (Ivanov et al., 2012).

# LjVAMP72a and LjVAMP72b Positively Controls the Elongation of Root Hairs

Suppression of LjVAMP72a and LjVAMP72b mRNAs also affected the elongation of root hairs. RNAi-LjVAMP72a/72b knockdown roots exhibited significantly fewer root hairs or almost root hair-less phenotypes (**Figure 6**), suggesting that LjVAMP72a and LjVAMP72b functioned not only in symbioses but also in the development of root hairs.

### Amino Acid Sequence Alignment Conserved for the Symbiosis

SNARE proteins have a highly conserved coined-coil domain, called a SNARE motif (Harbury, 1998). Uemura et al. (2004) found a six amino acid region that could be classified by its subcellular localization within the SNARE motif. **Figure 7** shows the sequence alignment of the VAMP72 groups. The aligned sequences were selected from legume plants, nonlegume plants including A. thaliana. The alignment shows that the six amino acid regions (RSQAQD and QFQADS) of VAMP72s in L. japonicus were identical to the AtVAMP72s that localize in plasma membrane and endosome, respectively. These common six amino acid regions were detected in other species: M. truncatula, A. thaliana, S. lycopersicum, and Vitis vulgaris. A different six amino acid region (HHQAQD) was conserved in the "symbiotic" VAMP72 subgroup, including LjVAMP72a and LjVAMP72b (**Figure 7**).

### DISCUSSION

For membrane trafficking, VAMP72 of R-SNARE is specific to green plants (Sanderfoot, 2007). Based on genome sequence information (Sato et al., 2008), rhizobia-induced LjVAMP72a and LjVAMP72b are classified in the symbiotic VAMP72 subgroup.

The producing knockdown mutant (RNAi-LjVAMP72a/72b) was suppressed both expressions (**Figure 5B**). In addition, RNS and AM were severely reduced on the LjVAMP72a/72b knockdown roots (**Figure 5**) and LjVAMP72a and LjVAMP72b were enriched in the symbiosome-containing fraction (**Figure 4F**), so LjVAMP72a and LjVAMP72b might be involved in the formation of a SM as reported in M. truncatula (Ivanov et al., 2012).

Both LjVAMP72a and LjVAMP72b were expressed in the nodules. The only difference was that the expression level of LjVAMP72a in the nodules was higher than that in the leaves or roots; however, LjVAMP72b was expressed constitutively in the leaves, roots, and nodules (**Figure 1**). A slight induction of LjVAMP72b appeared in the infected roots at 7 days compared with the non-infected roots. The data suggest that the expression ratio of LjVAMP72a and LjVAMP72b plays a role in forming the infection thread and the following SM. More data is needed to determine the different functions of the two.

Uemura et al. (2004) speculated that the six amino acid region within the SNARE motif differs depending on the subcellular localization in Arabidopsis VAMP72 proteins. From the genome data base, there were eight VAMP 72 genes in L. japonicus (**Figure 2**). In the alignment analysis (**Figure 7**), we could speculate the membrane localization of these Vamp72s from six amino acid region within the SNARE motif. Moreover, we found the symbiotic subgroup containing LjVAMP72a and LjVAMP72b conserved a different six amino acid region (HHQAQD). The symbiotic subgroup includes to non-legume genes, such as SlVAMP72a and VvVAMP72c (**Figure 2**, Ivanov et al., 2012) in S. lycopersicum and V. vulgaris, respectively, indicating that the VAMP72s with the six amino acid region (HHQAQD) is conserved among the R-SNARE for the localization of AM. The development of SM by LjVAMP72a and LjVAMP72b would be recruited during the evolution. The localization of another VAMP72s in L. japonicus (**Figure 7**) could be speculated from the six amino acid region that of AtVAMP72s localizing in the plasma membrane or endosome, except LjVAMP72f. The LjVAMP72f has a higher similarity to MtVAMP72c; however, there was no conserved amino acid in AtVAM72s of A. thaliana. Future studies may give more insight into the localization of LjVAMP72f.

We also found that LjVAMP72a/72b knockdown roots exhibited impaired root hair elongation (**Figure 6**), which was not reported in M. truncatula (Ivanov et al., 2012). Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized because of vesicle targeting (Enami et al., 2009). In Arabidopsis, VAMP721/722/724 localizes in root hairs, and these AtVAMP72s form SNARE complexes with Q-SNARE, SYP123, and SYP132 (Ichikawa et al., 2014). LjVAMP72a and LjVAMP72b belonging to VAMP721 would have dual functions of root hair development and symbiotic membrane formation. Therefore, the Q-SNAREs responsible for root hair development and symbiotic membrane formation need to be confirmed. Taken together, our data suggested that the LjVAMP72a and LjVAMP72b played a role in RNS and AM as well as root hair development. Dual physiological activity of LjVAMP72a and LjVAMP72b likely involves a change in the vesicle cargo during the transition from plant growth to the symbiosis stage.

# AUTHOR CONTRIBUTIONS

AS and AY performed most of the experiments and contributed equally. MH and MN designed the experiments. HY, MK, TM, and ST participated in some part of the study.

# FUNDING

This work was supported by a Grant-in-Aid (Grant No. 25450084, MN) and by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

### ACKNOWLEDGMENTS

fpls-09-01992 January 16, 2019 Time: 13:36 # 10

We thank Dr. Keisuke Yokota, National Institute of Agrobiological Sciences, Tsukuba, Japan and Dr. Shusei Sato, Tohoku University, Japan for their technical support.

### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01992/ full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Sogawa, Yamazaki, Yamasaki, Komi, Manabe, Tajima, Hayashi and Nomura. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Genetic Analysis and Mapping of QTLs for Soybean Biological Nitrogen Fixation Traits Under Varied Field Conditions

Qing Yang† , Yongqing Yang† , Ruineng Xu, Huiyong Lv and Hong Liao\*

Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, China

### Edited by:

Hon-Ming Lam, The Chinese University of Hong Kong, China

### Reviewed by:

Brett James Ferguson, The University of Queensland, Australia Oswaldo Valdes-Lopez, National Autonomous University of Mexico, Mexico Yinglong Chen, University of Western Australia, Australia

### \*Correspondence:

Hong Liao<EMAIL_ADDRESS><EMAIL_ADDRESS>
†These authors have contributed equally to this work

### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

Received: 16 August 2018 Accepted: 17 January 2019 Published: 01 February 2019

### Citation:

Yang Q, Yang Y, Xu R, Lv H and Liao H (2019) Genetic Analysis and Mapping of QTLs for Soybean Biological Nitrogen Fixation Traits Under Varied Field Conditions. Front. Plant Sci. 10:75. doi: 10.3389/fpls.2019.00075 Soybean is an important economic and green manure crop that is widely used in intercropping and rotation systems due to its high biological nitrogen fixation (BNF) capacity and the resulting reduction in N fertilization. However, the genetic mechanisms underlying soybean BNF are largely unknown. Here, two soybean parent genotypes contrasting in BNF traits and 168 F9:11 recombinant inbred lines (RILs) were evaluated under four conditions in the field. The parent FC1 always produced more big nodules, yet fewer nodules in total than the parent FC2 in the field. Furthermore, nodulation in FC1 was more responsive to environmental changes than that in FC2. Broad-sense heritability (h 2 b) for all BNF traits varied from 0.48 to 0.87, which suggests that variation in the observed BNF traits was primarily determined by genotype. Moreover, two new QTLs for BNF traits, qBNF-16 and qBNF-17, were identified in this study. The qBNF-16 locus was detected under all of the four tested conditions, where it explained 15.9– 59.0% of phenotypic variation with LOD values of 6.31–32.5. Meanwhile qBNF-17 explained 12.6–18.6% of observed variation with LOD values of 4.93–7.51. Genotype group analysis indicated that the FC1 genotype of qBNF-16 primarily affected nodule size (NS), while the FC2 genotype of qBNF-16 promoted nodule number (NN). On the other hand, the FC1 genotype of qBNF-17 influenced NN and the FC2 genotype of qBNF-17 impacted NS. The results on the whole suggest that these two QTLs might be valuable markers for breeding elite soybean varieties with high BNF capacities.

Keywords: QTLs (quantitative trait loci), BNF capacities, RILs (recombinant inbred lines), nodule number and nodule dry weight, nodule size, field conditions tests

# INTRODUCTION

As an important economic crop, soybean is a main source of edible oil and protein for human around the world due to the high oil (20–25%) and high protein (42–45%) contents in the seeds (Aziz et al., 2016). At the same time, the high capacity of biological nitrogen fixation (BNF) found in leguminous crops, including soybean, makes this a key source of green manure in agro-ecosystems (Kumudini et al., 2008; Chen and Liao, 2017; Yang et al., 2017).

Nitrogen (N) is one of the most limiting factors in crop production. In order to obtain high yields, farmers tend to supply excessive amounts of N fertilizers, which not only increases input costs, but also causes potentially adverse effects on the environment, including air and water

pollution (Santos et al., 2013; Li et al., 2016). Although N is abundant in the atmosphere, plants are unable to acquire it directly on their own, because it predominantly exists in the inert form of N2. BNF is a process in which plant unavailable atmospheric N2 is converted into readily available ammonia (NH3) in nodules formed through symbiotic associations between plants and microbes (Fox et al., 2016; Yang et al., 2017).

Chemical synthesis provides 118 million metric tons of fertilizer N each year (Joseph et al., 2016). In comparison, it is estimated that symbiosis between nitrogen-fixing rhizobia and plants provides 50–70 million tons of N for agricultural systems each year (Herridge et al., 2008). As one of the most important legume crops, soybean fixes 16.4 million tons of N annually, which represents 77% of the total N fixed by legume crops (Herridge et al., 2008). In modern agricultural systems, soybean is typically considered to have the most potential for sustaining green agricultural systems. For example, in Brazil, over 70% of the N required for soybean growth is derived primarily from BNF (Peoples et al., 2009). Therefore, breeding elite soybean cultivars with high BNF capacities and high yields could be an efficient way to maintain agriculture sustainability.

Soybean BNF capacity is influenced by many environmental factors. Among them, rhizobial strains play critically roles in the nitrogen fixation capacity of soybean nodules (Denison and Kiers, 2004), and which could be classed into three genotype/phenotypes: mutualistic rhizobia, parasitic rhizobia and non-symbiotic. To facilitate agriculture sustainability, mutualistic rhizobia are preferred to become parasitic and nonsymbiotic rhizobia, because that parasitic and non-symbiotic rhizobia fix little nitrogen or even unable to infect legumes at all (Denison and Kiers, 2004). For example, Yang et al. (2017) found that upon inoculation with effective rhizobial strains, nodule numbers and dry weights of a RIL population did not significantly vary compared to inoculation with less effective strains, yet shoot dry weight increased by 18.25% on average. Other research has revealed that inoculation with effective rhizobium strains in soybean not only enhances nodule fresh weight, but also increases N and P contents, as well as, yield in the field (Qin et al., 2012). These results imply that rhizobial inoculation increases soybean BNF capacity mainly due to the effects of particular rhizobial species and strains.  Furthermore, water stress also appears to affect BNF capacity (Nascimento et al., 2016; Jemo et al., 2017). In short, BNF is a complex process that is affected by host and symbiotic genotypes, along with the context of environmental conditions in which the symbiosis occurs.

High BNF efficiency mainly depends on the phenotypes expressed in symbiosis between host plants and rhizobia (Nicolás et al., 2006), including the readily observable traits of nodule number (NN), nodule weight (NW), and nodule size (NS). In a relatively stable field environment, the heritability of nodulation traits may exceed 0.8 (Yang et al., 2017), suggesting that nodulation traits are mainly controlled by genetic loci. To date, a number of QTLs for nodulation traits have been identified at different stages of soybean development in pots or in field.  Some QTLs, such as qBNF-C2, qBNF-O and qBNF-B1, are co-located with yield trait loci (Yang et al., 2017), implying that BNF capacity might affect soybean yield. Therefore, breeding new soybean varieties that produce higher yields through optimization of BNF capacity promises to be a feasible and economic strategy. Unfortunately, possibly due to technical limitations and labor costs, most of the QTLs for BNF traits obtained from field observations are preliminary and minor, so they are not likely to be useful as markers in soybean breeding programs. As a result, progress in studying BNF associated QTL markers in field experiments has lagged behind marker studies for other soybean traits.