Journal of Plant Physiology 171 (2014) 104–108
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Molecular Biology
Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus Sirinapa Chungopast a,b , Pilunthana Thapanapongworakul a,c , Hiroyuki Matsuura a , Tan Van Dao a,d , Toshimasa Asahi a , Kuninao Tada a , Shigeyuki Tajima a , Mika Nomura a,∗ a
Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakorn Pathom 73140, Thailand c Faculty of Agriculture, Chiang Mai University, 50200 Chiang Mai, Thailand d Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy Road, Hanoi, Vietnam b
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 25 October 2013 Accepted 25 October 2013 Available online 21 December 2013 Keywords: Glutamine synthetase Mesorhizobium loti Nodule Senescence
a b s t r a c t In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume–rhizobia symbiosis by modifying nodule development and metabolic processes. © 2013 Elsevier GmbH. All rights reserved.
Introduction Nitrogen fixation by rhizobium–legume symbiosis provides a significant proportion of the available nitrogen in the biosphere, making this relationship agronomically and ecologically important. Ammonium is the primary stable product of nitrogen fixation, in which bacteroids secrete ammonium into the plant, and it is assimilated into glutamate by glutamine synthetase (GS) and glutamate synthase (GOGAT) (Cullimore and Bennett, 1988; Temple et al., 1998). Bacteroids simply provide the plant with ammonium because the major assimilating pathways through GS and GOGAT are repressed in bacteroids (Brown and Dilworth, 1975; Kurz et al., 1975; Cullimore and Bennett, 1991; Temple et al., 1998). However, the nitrogen nutrient exchange between the plant cytosol and bacteroids is more complex. Recently, an amino-acid cycle has been reported to be essential for symbiotic nitrogen fixation by rhizobium in pea nodules (Lodwig et al., 2003; Prell et al., 2009). Given the importance of amino acid transport into bacteroids to support N2 fixation, understanding the role of the endogenous synthesis of amino acids inside bacteroids by the
Abbreviations: ARA, acetylene reduction activity; GS, glutamine synthetase; STM, signature-tagged mutagenesis; wpi, weeks post-inoculation; WT, wild-type. ∗ Tel.: +81 87 891 3135; fax: +81 87 891 3021. E-mail address: [email protected] (M. Nomura). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.015
GS/GOGAT is important. GOGAT and GSI/GSII double mutants of Bradyrhizobium japonicum are Fix− , whereas GOGAT and GSI/GSII double mutants of Sinorhizobium meliloti are Fix+ (Osburne and Signer, 1980; O’Gara et al., 1984; Carlson et al., 1987; deBruijn et al., 1989; Lewis et al., 1990). These results clearly indicate that B. japonicum needs to make glutamine and/or glutamate to form effective Fix+ nodules, and S. meliloti does not. However, there is no report of the function of ammonium assimilation in Mesorhizobium loti and Lotus japonicus symbiosis. To understand the role of ammonium assimilation in N2 fixation by legume bacteroids, mutating the GS/GOGAT pathway is essential. On the RhizoBase website (http://genome.microbedb.jp/rhizobase/Mesorhizobium), we found nine proposed GS genes in M. loti MAFF30309: mll0343 (glutamine synthetase I, GSI), mlr0339 (glutamine synthetase II, GSII), mll7307 (glutamine synthetase III, GSIII), mlr6210 (GSIII), mll5148, mll3074, mll7254, mll6521, and mll4187. To understand the function of ammonium assimilation in more detail, we used a rhizobial mutant produced using a signature-tagged mutagenesis (STM) technique. Shimoda et al. (2008) constructed a large-scale random mutagenesis of M. loti using STM. STM is based on transposon insertional mutagenesis that allows for large numbers of mutants to be analyzed simultaneously. This analysis is accomplished by tagging each mutant with a unique short DNA sequence to subsequently distinguish different mutants with unique signature tags. Sequencing the transposon insertion sites enabled the collection of defined sets of transposon mutants.
S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
105
A mlr0346
mll0345
msr0341
mlr0339
mll0343
0.1kb EcoRI
PstI
SphI
Tn5
Relative expression
B 100 90 80 70 60 50 40 30 20 10 0
* WT
STM30 Strain
Fig. 1. The STM30 transposon insertion mutant for M. loti and GSI expression in the bacteroids. A transposon (Tn5) was inserted in the glutamine synthetase I (GSI, mll0343) gene (A). Each RNA was isolated from the bacteroids to synthesize the cDNA and then qRT-PCR was performed (B). The expression was normalized to sigA. All of the data are shown as ±SD. Statistically significant differences compared with the wild-type bacteroid are indicated with * (P < 0.01).
These mutants were identified using the RhizoBase database. We searched various GS mutants from the database and found that the STM30 mutant had a transposon inserted in the GSI (mll0343) gene (Fig. 1). In this study, we focused on GSI in M. loti and found that GSI (mll0343) deficiency in M. loti induced early nodule senescence. Materials and methods Bacterial strains and medium The Mesorhizobium loti MAFF303099, a gram-negative, nitrogen-fixing, symbiotic bacterium for Lotus japonicus, was used in this study. The transposon insertion mutant strain of M. loti, STM30, was generated using the signature-tagged mutagenesis (STM) technique described by Shimoda et al. (2008). STM30 had a Tn inserted at base pair 399 of the 1410-bp GSI gene (mll0343; Fig. 1A). The M. loti strain M. loti strains were cultured in tryptone-yeast extract (TY) liquid medium (Beringer, 1974) at 28 ◦ C.
Assay for acetylene reduction To assay the nitrogenase activity in the nodules, the acetylene reduction activity (ARA) in vivo was measured. Intact whole plants bearing nodules or collected nodules were placed in a 70-mL glass vial and incubated at 25 ◦ C with 7 mL of acetylene. After a 30min incubation, the amount of ethylene produced was measured by gas chromatography (GC-8A Shimadzu) as previously described (Suganuma et al., 1998). Free amino acid measurement For the amino acid analysis, the bacteroids were isolated from L. japonicus nodules as previously reported (Kouchi et al., 1991; Hoa et al., 2004). The nodules, leaves and isolated bacteroids were ground with a mortar and pestle in 80% ethanol, respectively. Then, the samples were heated to 80 ◦ C, dried, and suspended in 0.02 M HCl. The amino acids were analyzed using a Hitachi amino acid analyzer (L-8900BH, Hitachi). RNA extraction and quantitative real-time PCR
Plant growth conditions Seeds from L. japonicus GIFU (Borjigin et al., 2011) were scarified using sandpaper to remove the seed coats. Then, 1% sodium hypochlorite solution containing 0.01% Tween-20 was used to surface sterilize the seeds for 10 min. Vermiculite was placed in the upper compartments of Magenta Jars containing a half-diluted B&D nitrogen-free nutrient solution (Broughton and Dilworth, 1971). Twenty-five seeds were cultured in each jar and germinated under sterile conditions. The jars were placed in a growth cabinet (EYELA FLI-2000) at 24 ◦ C and exposed to 16-h/light and 8-h/dark cycles. After seven days, the plants were inoculated with 10 mL of M. loti (1 × 109 cells mL−1 ). These plants were examined at one, two, four, and seven weeks post-inoculation (wpi).
The bacteroids were isolated from the nodules at 4 wpi as described previously (Hoa et al., 2004). The RNA from the bacteroids was isolated using the RNAwiz (Life Technologies) and then purified using RNeasy spin columns (Qiagen). For the RNA extraction from the nodules, 0.1 g of the nodules were ground with a mortar and pestle followed by total RNA isolation using the RNeasy Plant Mini Kit (Qiagen). Each RNA was converted to cDNA using the PrimeScript 1st strand cDNA synthesis kit (Takara). The resulting cDNA was amplified with specific primer pairs (0343Fw, 5 -CGACCAGATCGACAGCTACA-3 ; 0343Rv, 5 -ATTTCAAAGCGCATCACCTC-3 ; sigAFw, 5 -CATCTCCATCGCCAAGAAAT-3 ; sigARv, 5 -GAACTTATCGACCGCCTTCA-3 ; LjCysFw, 5 -GAG CAG ATT GGG GTG AAG AA-3 ; CysRv, 5 -ATC CAC ATC CCT TTG CAT TC-3 ; Ubi Fw, 5 -ATG CAG
106
S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
ATC TTC GTC AAG ACC TTG-3 ; UbiRv, 5 -ACC TCC CCT CAG ACG AAG-3 ; LjLbFw, 5 -TTT GAG CAC TGC TTG GGG AGT AGC T-3 ; and LjLbRv, 5 -AGG CAT GCA AAA CCA GAA AC-3 ) and the Takara SYBR Green premix ExTaq II (Takara). Then, sigA (sigAFw, sigARv) was used as an internal reference for the relative quantification of the bacteroids. The ubiquitin transcript (UbiFw, UbiRv) was used as an internal reference for the nodules. The quantitative real-time PCRs were performed using the Thermal Cycler Dice Real-time System II (Takara). Chlorophyll analysis To measure the concentration of chlorophyll, the leaves were ground with 100% EtOH using a mortar and pestle and then centrifuged. The supernatant was measured at absorbances of 730, 665 and 649 nm as described by Shen et al. (2009). Results Quantitative real-time PCR and growth phenotype of STM30 The GSI mutant, STM30, has a Tn insertion in the GSI gene (mll0343), which was prepared by Shimoda et al. (2008) (Fig. 1A). To determine the GSI expression level in the STM30 bacteroids, we performed quantitative real-time PCR. Fig. 1B shows that the expression of mll0343 GSI in the bacteroids isolated from STM30 was significantly lower than the bacteroids isolated from the wildtype (WT) strain. This result indicates that STM30 is a GSI-deficient mutant. Effect of GS deficiency in bacteroids To test the nodulation ability of STM30, the germinated seeds were inoculated with STM30 or the WT, M. loti MAFF303099, as a control. The plants were inoculated one week post-germination and grown in Magenta jars for nine more weeks during which the nodule number and color were determined (Fig. 2). These plants exhibited significant differences in nodule number during the nine weeks of growth. The number of nodules for the plants inoculated with STM30 continued to increase linearly for nine weeks after inoculation, whereas the number of nodules for the plants with the WT M. loti reached a plateau (Fig. 2A). The plants with the WT M. loti had pink nodules starting approximately two weeks postinoculation (wpi), and the color did not change at 4 wpi. However, many nodules inoculated with STM30 showed a senescence phenotype (green color nodule) beginning at 4 wpi (Fig. 2B). To assess the senescence phenotype, we monitored the color changes in the nodules over a 9-week period for the plants inoculated with STM30 or the WT M. loti. The macroscopic indicator of nodule senescence is a color shift in the nodule from pink to green (Fig. 2C). This color shift is associated with the degradation of plant leghemoglobin, and the nitrogenase activity of the green nodules was negligible (data not shown). The first signs of senescence appeared at 4 wpi in the nodules inoculated with STM30. The nodules inoculated with STM30 accelerated the senescence drastically during the nine weeks of growth, and 50% of the nodules appeared green at 7 wpi, whereas only 20% of the nodules with WT M. loti were green (Fig. 2B). Although the nodules inoculated with STM30 displayed early senescence, the apparent acetylene reduction activity (ARA) for the STM30 plants was similar to the plants with WT M. loti. However, when we calculated the ARA on a per nodule basis, the ARA of the STM30-inoculated nodules was significantly lower than the WT-inoculated nodules (Fig. 3A). We also measured the nodule diameter and found that the size of the nodules inoculated with STM30 was smaller than the WT-inoculated nodules (Fig. 3B).
Fig. 2. Nodulation rate and phenotype of the plants inoculated with the wild-type M. loti (WT) and STM30. Nodule number per plant (A) and the ratio of green nodules to the total number of nodules (B) were examined nine weeks post-inoculation (wpi). The color of the nodules inoculated with the wild-type M. loti (pink nodule) and STM30 (pink and green nodules) at four weeks post-inoculation were determined (C). All of the data are shown as ±SD. *A significant difference (P < 0.01). Bar = 200 m.
These data suggest that the nodules inoculated with STM30 rapidly senesced, leading to the production of more nodules. Expression of leghemoglobin and cysteine protease in the nodules Cysteine protease activity has been reported in senescent nodules from French beans (Pladys and Rigaud, 1985). To prove that the nodules inoculated with the GSI mutant underwent early senescence, we measured the relative expression of the cysteine protease gene in the nodules using quantitative RT-PCR and gene-specific primers (Fig. 4). A different expression pattern was observed from 4 wpi (Fig. 4A). Cysteine protease expression in the STM30-inoculated nodules was considerably higher than the WT-inoculated nodules. The cysteine proteases have been believed to degrade leghemoglobin, which is essential for maintaining the
5.0
A
4.0
STM30 WT
3.0
**
2.0
*
1.0 0.0 0
2
4 6 8 Weeks post inoculation
Nodule diameter (mm)
0.8 0.6
2.0 1.5 1.0
4
7
0.4 0.2
Strains
STM30
70 Relative expression
**
2.5
Fig. 5. The chlorophyll content in the leaves from the L. japonicus plants inoculated with the wild-type M. loti ( ) or STM30 () at four and seven weeks post-inoculation. The chlorophyll was extracted using 100% EtOH and measured at 730, 665 and 649 nm as described by Shen et al. (2009). All of the data are shown as ±SD. * and ** indicate a significant difference (*P < 0.05; **P < 0.01).
**
1.0
Fig. 3. Acetylene reduction activity and diameter of the nodules inoculated with the wild-type M. loti (WT) or STM30. The acetylene reduction activity per nodule (A) was measured 9 wpi. The diameters of the nodules (B) at 4 wpi are shown. All of the data are shown as ±SD. * and ** indicate a significant difference (*P < 0.05; **P < 0.01).
**
A
50 40 30
**
20 10 0 4
7 Weeks post inoculation
40
Relative expression
*
3.0
Weeks post inoculation
WT
35
3.5
0.0
B
0.0
60
4.0
0.5
10
1.4 1.2
107
4.5
*
Chlorophyll concentration (mg Chl gFw-1)
Acetylene reduction activity (nmol h -1 nodule-1)
S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
B
nitrogen-fixing activity of the nodules (Pladys et al., 1991). When we measured leghemoglobin gene expression in the nodules, the STM30-inoculated nodules had reduced expression compared with the WT-inoculated nodules (Fig. 4B). These data strongly support the hypothesis that STM30 inoculation induces early nodule senescence. Characteristics of the plants inoculated with STM30 or WT M. loti When the nodules inoculated with STM30 became greener, the leaf color appeared yellowish (Fig. 5). When we measured the chlorophyll content in the leaves, the values for the plants inoculated with STM30 was 35% lower than the leaves from the WT-inoculated plants at 7 wpi. These data suggest that the GSI in the bacteroids may be detrimental to the symbiosis. Nitrogen-fixing plants can be classified as amide or ureide exporters based on the composition of the xylem fluid collected from the excised nodules (Schubert, 1986). The temperate legume L. japonicus exports asparagine or glutamine out of the nodule to the shoot as the major products of N2 fixation (Schubert, 1986). We measured the free amino acid content of glutamine, asparagine, glutamate and aspartate in the bacteroids, nodules and leaves at 7 wpi (Table 1). Remarkably, the concentration of free amino acids, especially glutamate, glutamine and asparagine, in the bacteroids, nodules and leaves inoculated with STM30 were lower compared with the plants inoculated with the WT M. loti. These data suggest that the legume–rhizobia symbiosis is severely affected by STM30.
30
Discussion
25 20
**
15
*
10 5 0 4
7 Weeks post inoculation
Fig. 4. Expression patterns of the cysteine protease (A) and leghemoglobin (B) genes in the nodules inoculated with STM30 () or wild-type ( ) M. loti. The qRT-PCR analysis was performed using the cDNA from the nodules harvested at four and seven weeks post-inoculation. The expression was normalized to ubiquitin. All of the data are shown as ±SD. Statistically significant differences compared with the wild-type nodules are indicated with * (P < 0.05) or ** (P < 0.01).
STM30 is a mutant strain containing a Tn insertion in the GSI gene (Fig. 1A). Although there are nine GS family genes in the M. loti genome, there is only one GSI gene (mll0343). When we produced other GS mutants by inserting a Tn in each gene (mlr0339, mlr6210, mll7254, mll7307, mll5148, and mll6521), we could not detect any nodule senescence, except for the STM30 mutant (data not shown). The nodule number per plant infected with STM30 increased continuously throughout the growth period (Fig. 2A). Many of the nodules for the plants infected with STM30 carried senesced green nodules (Fig. 2B). The ARA per STM30-inoculated nodule was significantly lower compared with the nodules inoculated with WT M. loti (Fig. 3A). As a biochemical indicator of nodule senescence, cysteine protease activity for degrading leghemoglobin has been reported
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S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
Table 1 The free amino acids in bacteroids and leaves of L. japonicus inoculated with wild-type or STM30 mutant. Values are the mean ± standard deviations (n = 5). Statistical significant differences compared with wild-type are indicated with * (P < 0.05) and ** (P < 0.01). Free amino acid
Nodules
Bacteroids
Wild-type −1
(nmol gFw Glu Gln Asn Asp
STM30
Wild-type
Leaves STM30
Wild-type
STM30
) 602.37 39.22 2350.87 61.55
± ± ± ±
105.75 8.66 262.16 15.53
274.52 21.98 1315.73 51.84
± ± ± ±
125.02* 6.91* 186.75** 21.16
26.67 1.36 41.11 3.55
(Pladys and Rigaud, 1985; Pladys et al., 1991). Early nodule senescence has shown to lead to a higher expression of cysteine protease genes (Pladys and Vance, 1993). After inoculation with STM30, many green nodules, apparently lacking intact leghemoglobin, appeared; in addition, cysteine protease expression increased (Fig. 4A), whereas leghemoglobin expression decreased (Fig. 4B). B. japonicum glnA or glnB single mutants have been reported to produce an increased number of nodules (Carlson et al., 1987). However, the reason for this increase in nodule number was not clear. We speculate that the B. japonicum glnA single mutant might also induce early nodule senescence. Taken together, these data suggest that the nodules inoculated with STM30 had reduced nitrogen-fixing activity per nodule and early nodule senescence. To maintain the nitrogen fixing activity, the plants inoculated with STM30 might produce many nodules. Early nodule senescence also induces plant senescence. The chlorophyll concentration in the leaves of STM30-infected plants was lower compared with the leaves of the WT infected plants. Temperate legumes, such as L. japonicus, pea, clover, and alfalfa, mainly export glutamine or asparagine out of the nodules to the shoot as a nitrogen compound (Temple et al., 1998; Goggin et al., 2003). The concentration of free amino acids, such as glutamine and asparagine, in the leaves of STM30-infected plants was lower (Table 1). The bacteroids with GSI deficiency did not fix nitrogen effectively and might not produce enough nitrogen compounds, such as glutamine and asparagine, in the nodules (Fig. 3 and Table 1). The lower chlorophyll concentration in the plants inoculated with STM30 might be the reason that glutamine and asparagine could not be effectively exported to the shoot. In recent years, one of the two amino acid uptake systems (Aap and Bra) in Rhizobium leguminosarum has been reported to be essential for symbiotic nitrogen fixation in pea nodules (Lodwig et al., 2003). In the absence of an amino acid transport system between the bacteroids and the plant cytoplasm, the bacteroids cannot efficiently fix nitrogen. Another report has indicated that an increase in the intracellular glutamine/glutamate ratio triggers the post-transcriptional downregulation of amino acid uptake by Aap and Bra (Mulley et al., 2011). The glutamine/glutamate ratio in bacteroids inoculated with STM30 was two-fold higher compared with nodules with the WT M. loti (Table 1), suggesting that STM30 also might suppress amino acid transport via Aap/Bra. Thus, we conclude from these data that the GSI in bacteroids plays an important role in maintaining legume–rhizobia symbiosis. A biochemical survey of the role of GSI in bacteroids for protecting nodules from senescence is underway in our laboratory. Acknowledgments This study was supported by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank the National BioResource Project for the Mesorhizobium loti STM30 mutant (STM clone ID, 10T05g06). C.S. was supported by Thailand government scholarship.
± ± ± ±
0.41 0.16 8.01 0.86
8.47 0.95 25.67 3.80
± ± ± ±
1.03** 0.08** 3.22** 0.34
1198.70 76.31 1239.90 296.52
± ± ± ±
112.24 2.92 70.53 15.10
944.05 41.51 592.61 283.95
± ± ± ±
72.73** 8.09** 24.19** 3.28
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Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Molecular Biology
Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus Sirinapa Chungopast a,b , Pilunthana Thapanapongworakul a,c , Hiroyuki Matsuura a , Tan Van Dao a,d , Toshimasa Asahi a , Kuninao Tada a , Shigeyuki Tajima a , Mika Nomura a,∗ a
Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakorn Pathom 73140, Thailand c Faculty of Agriculture, Chiang Mai University, 50200 Chiang Mai, Thailand d Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy Road, Hanoi, Vietnam b
a r t i c l e
i n f o
Article history: Received 2 August 2013 Received in revised form 25 October 2013 Accepted 25 October 2013 Available online 21 December 2013 Keywords: Glutamine synthetase Mesorhizobium loti Nodule Senescence
a b s t r a c t In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume–rhizobia symbiosis by modifying nodule development and metabolic processes. © 2013 Elsevier GmbH. All rights reserved.
Introduction Nitrogen fixation by rhizobium–legume symbiosis provides a significant proportion of the available nitrogen in the biosphere, making this relationship agronomically and ecologically important. Ammonium is the primary stable product of nitrogen fixation, in which bacteroids secrete ammonium into the plant, and it is assimilated into glutamate by glutamine synthetase (GS) and glutamate synthase (GOGAT) (Cullimore and Bennett, 1988; Temple et al., 1998). Bacteroids simply provide the plant with ammonium because the major assimilating pathways through GS and GOGAT are repressed in bacteroids (Brown and Dilworth, 1975; Kurz et al., 1975; Cullimore and Bennett, 1991; Temple et al., 1998). However, the nitrogen nutrient exchange between the plant cytosol and bacteroids is more complex. Recently, an amino-acid cycle has been reported to be essential for symbiotic nitrogen fixation by rhizobium in pea nodules (Lodwig et al., 2003; Prell et al., 2009). Given the importance of amino acid transport into bacteroids to support N2 fixation, understanding the role of the endogenous synthesis of amino acids inside bacteroids by the
Abbreviations: ARA, acetylene reduction activity; GS, glutamine synthetase; STM, signature-tagged mutagenesis; wpi, weeks post-inoculation; WT, wild-type. ∗ Tel.: +81 87 891 3135; fax: +81 87 891 3021. E-mail address: [email protected] (M. Nomura). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.015
GS/GOGAT is important. GOGAT and GSI/GSII double mutants of Bradyrhizobium japonicum are Fix− , whereas GOGAT and GSI/GSII double mutants of Sinorhizobium meliloti are Fix+ (Osburne and Signer, 1980; O’Gara et al., 1984; Carlson et al., 1987; deBruijn et al., 1989; Lewis et al., 1990). These results clearly indicate that B. japonicum needs to make glutamine and/or glutamate to form effective Fix+ nodules, and S. meliloti does not. However, there is no report of the function of ammonium assimilation in Mesorhizobium loti and Lotus japonicus symbiosis. To understand the role of ammonium assimilation in N2 fixation by legume bacteroids, mutating the GS/GOGAT pathway is essential. On the RhizoBase website (http://genome.microbedb.jp/rhizobase/Mesorhizobium), we found nine proposed GS genes in M. loti MAFF30309: mll0343 (glutamine synthetase I, GSI), mlr0339 (glutamine synthetase II, GSII), mll7307 (glutamine synthetase III, GSIII), mlr6210 (GSIII), mll5148, mll3074, mll7254, mll6521, and mll4187. To understand the function of ammonium assimilation in more detail, we used a rhizobial mutant produced using a signature-tagged mutagenesis (STM) technique. Shimoda et al. (2008) constructed a large-scale random mutagenesis of M. loti using STM. STM is based on transposon insertional mutagenesis that allows for large numbers of mutants to be analyzed simultaneously. This analysis is accomplished by tagging each mutant with a unique short DNA sequence to subsequently distinguish different mutants with unique signature tags. Sequencing the transposon insertion sites enabled the collection of defined sets of transposon mutants.
S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
105
A mlr0346
mll0345
msr0341
mlr0339
mll0343
0.1kb EcoRI
PstI
SphI
Tn5
Relative expression
B 100 90 80 70 60 50 40 30 20 10 0
* WT
STM30 Strain
Fig. 1. The STM30 transposon insertion mutant for M. loti and GSI expression in the bacteroids. A transposon (Tn5) was inserted in the glutamine synthetase I (GSI, mll0343) gene (A). Each RNA was isolated from the bacteroids to synthesize the cDNA and then qRT-PCR was performed (B). The expression was normalized to sigA. All of the data are shown as ±SD. Statistically significant differences compared with the wild-type bacteroid are indicated with * (P < 0.01).
These mutants were identified using the RhizoBase database. We searched various GS mutants from the database and found that the STM30 mutant had a transposon inserted in the GSI (mll0343) gene (Fig. 1). In this study, we focused on GSI in M. loti and found that GSI (mll0343) deficiency in M. loti induced early nodule senescence. Materials and methods Bacterial strains and medium The Mesorhizobium loti MAFF303099, a gram-negative, nitrogen-fixing, symbiotic bacterium for Lotus japonicus, was used in this study. The transposon insertion mutant strain of M. loti, STM30, was generated using the signature-tagged mutagenesis (STM) technique described by Shimoda et al. (2008). STM30 had a Tn inserted at base pair 399 of the 1410-bp GSI gene (mll0343; Fig. 1A). The M. loti strain M. loti strains were cultured in tryptone-yeast extract (TY) liquid medium (Beringer, 1974) at 28 ◦ C.
Assay for acetylene reduction To assay the nitrogenase activity in the nodules, the acetylene reduction activity (ARA) in vivo was measured. Intact whole plants bearing nodules or collected nodules were placed in a 70-mL glass vial and incubated at 25 ◦ C with 7 mL of acetylene. After a 30min incubation, the amount of ethylene produced was measured by gas chromatography (GC-8A Shimadzu) as previously described (Suganuma et al., 1998). Free amino acid measurement For the amino acid analysis, the bacteroids were isolated from L. japonicus nodules as previously reported (Kouchi et al., 1991; Hoa et al., 2004). The nodules, leaves and isolated bacteroids were ground with a mortar and pestle in 80% ethanol, respectively. Then, the samples were heated to 80 ◦ C, dried, and suspended in 0.02 M HCl. The amino acids were analyzed using a Hitachi amino acid analyzer (L-8900BH, Hitachi). RNA extraction and quantitative real-time PCR
Plant growth conditions Seeds from L. japonicus GIFU (Borjigin et al., 2011) were scarified using sandpaper to remove the seed coats. Then, 1% sodium hypochlorite solution containing 0.01% Tween-20 was used to surface sterilize the seeds for 10 min. Vermiculite was placed in the upper compartments of Magenta Jars containing a half-diluted B&D nitrogen-free nutrient solution (Broughton and Dilworth, 1971). Twenty-five seeds were cultured in each jar and germinated under sterile conditions. The jars were placed in a growth cabinet (EYELA FLI-2000) at 24 ◦ C and exposed to 16-h/light and 8-h/dark cycles. After seven days, the plants were inoculated with 10 mL of M. loti (1 × 109 cells mL−1 ). These plants were examined at one, two, four, and seven weeks post-inoculation (wpi).
The bacteroids were isolated from the nodules at 4 wpi as described previously (Hoa et al., 2004). The RNA from the bacteroids was isolated using the RNAwiz (Life Technologies) and then purified using RNeasy spin columns (Qiagen). For the RNA extraction from the nodules, 0.1 g of the nodules were ground with a mortar and pestle followed by total RNA isolation using the RNeasy Plant Mini Kit (Qiagen). Each RNA was converted to cDNA using the PrimeScript 1st strand cDNA synthesis kit (Takara). The resulting cDNA was amplified with specific primer pairs (0343Fw, 5 -CGACCAGATCGACAGCTACA-3 ; 0343Rv, 5 -ATTTCAAAGCGCATCACCTC-3 ; sigAFw, 5 -CATCTCCATCGCCAAGAAAT-3 ; sigARv, 5 -GAACTTATCGACCGCCTTCA-3 ; LjCysFw, 5 -GAG CAG ATT GGG GTG AAG AA-3 ; CysRv, 5 -ATC CAC ATC CCT TTG CAT TC-3 ; Ubi Fw, 5 -ATG CAG
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ATC TTC GTC AAG ACC TTG-3 ; UbiRv, 5 -ACC TCC CCT CAG ACG AAG-3 ; LjLbFw, 5 -TTT GAG CAC TGC TTG GGG AGT AGC T-3 ; and LjLbRv, 5 -AGG CAT GCA AAA CCA GAA AC-3 ) and the Takara SYBR Green premix ExTaq II (Takara). Then, sigA (sigAFw, sigARv) was used as an internal reference for the relative quantification of the bacteroids. The ubiquitin transcript (UbiFw, UbiRv) was used as an internal reference for the nodules. The quantitative real-time PCRs were performed using the Thermal Cycler Dice Real-time System II (Takara). Chlorophyll analysis To measure the concentration of chlorophyll, the leaves were ground with 100% EtOH using a mortar and pestle and then centrifuged. The supernatant was measured at absorbances of 730, 665 and 649 nm as described by Shen et al. (2009). Results Quantitative real-time PCR and growth phenotype of STM30 The GSI mutant, STM30, has a Tn insertion in the GSI gene (mll0343), which was prepared by Shimoda et al. (2008) (Fig. 1A). To determine the GSI expression level in the STM30 bacteroids, we performed quantitative real-time PCR. Fig. 1B shows that the expression of mll0343 GSI in the bacteroids isolated from STM30 was significantly lower than the bacteroids isolated from the wildtype (WT) strain. This result indicates that STM30 is a GSI-deficient mutant. Effect of GS deficiency in bacteroids To test the nodulation ability of STM30, the germinated seeds were inoculated with STM30 or the WT, M. loti MAFF303099, as a control. The plants were inoculated one week post-germination and grown in Magenta jars for nine more weeks during which the nodule number and color were determined (Fig. 2). These plants exhibited significant differences in nodule number during the nine weeks of growth. The number of nodules for the plants inoculated with STM30 continued to increase linearly for nine weeks after inoculation, whereas the number of nodules for the plants with the WT M. loti reached a plateau (Fig. 2A). The plants with the WT M. loti had pink nodules starting approximately two weeks postinoculation (wpi), and the color did not change at 4 wpi. However, many nodules inoculated with STM30 showed a senescence phenotype (green color nodule) beginning at 4 wpi (Fig. 2B). To assess the senescence phenotype, we monitored the color changes in the nodules over a 9-week period for the plants inoculated with STM30 or the WT M. loti. The macroscopic indicator of nodule senescence is a color shift in the nodule from pink to green (Fig. 2C). This color shift is associated with the degradation of plant leghemoglobin, and the nitrogenase activity of the green nodules was negligible (data not shown). The first signs of senescence appeared at 4 wpi in the nodules inoculated with STM30. The nodules inoculated with STM30 accelerated the senescence drastically during the nine weeks of growth, and 50% of the nodules appeared green at 7 wpi, whereas only 20% of the nodules with WT M. loti were green (Fig. 2B). Although the nodules inoculated with STM30 displayed early senescence, the apparent acetylene reduction activity (ARA) for the STM30 plants was similar to the plants with WT M. loti. However, when we calculated the ARA on a per nodule basis, the ARA of the STM30-inoculated nodules was significantly lower than the WT-inoculated nodules (Fig. 3A). We also measured the nodule diameter and found that the size of the nodules inoculated with STM30 was smaller than the WT-inoculated nodules (Fig. 3B).
Fig. 2. Nodulation rate and phenotype of the plants inoculated with the wild-type M. loti (WT) and STM30. Nodule number per plant (A) and the ratio of green nodules to the total number of nodules (B) were examined nine weeks post-inoculation (wpi). The color of the nodules inoculated with the wild-type M. loti (pink nodule) and STM30 (pink and green nodules) at four weeks post-inoculation were determined (C). All of the data are shown as ±SD. *A significant difference (P < 0.01). Bar = 200 m.
These data suggest that the nodules inoculated with STM30 rapidly senesced, leading to the production of more nodules. Expression of leghemoglobin and cysteine protease in the nodules Cysteine protease activity has been reported in senescent nodules from French beans (Pladys and Rigaud, 1985). To prove that the nodules inoculated with the GSI mutant underwent early senescence, we measured the relative expression of the cysteine protease gene in the nodules using quantitative RT-PCR and gene-specific primers (Fig. 4). A different expression pattern was observed from 4 wpi (Fig. 4A). Cysteine protease expression in the STM30-inoculated nodules was considerably higher than the WT-inoculated nodules. The cysteine proteases have been believed to degrade leghemoglobin, which is essential for maintaining the
5.0
A
4.0
STM30 WT
3.0
**
2.0
*
1.0 0.0 0
2
4 6 8 Weeks post inoculation
Nodule diameter (mm)
0.8 0.6
2.0 1.5 1.0
4
7
0.4 0.2
Strains
STM30
70 Relative expression
**
2.5
Fig. 5. The chlorophyll content in the leaves from the L. japonicus plants inoculated with the wild-type M. loti ( ) or STM30 () at four and seven weeks post-inoculation. The chlorophyll was extracted using 100% EtOH and measured at 730, 665 and 649 nm as described by Shen et al. (2009). All of the data are shown as ±SD. * and ** indicate a significant difference (*P < 0.05; **P < 0.01).
**
1.0
Fig. 3. Acetylene reduction activity and diameter of the nodules inoculated with the wild-type M. loti (WT) or STM30. The acetylene reduction activity per nodule (A) was measured 9 wpi. The diameters of the nodules (B) at 4 wpi are shown. All of the data are shown as ±SD. * and ** indicate a significant difference (*P < 0.05; **P < 0.01).
**
A
50 40 30
**
20 10 0 4
7 Weeks post inoculation
40
Relative expression
*
3.0
Weeks post inoculation
WT
35
3.5
0.0
B
0.0
60
4.0
0.5
10
1.4 1.2
107
4.5
*
Chlorophyll concentration (mg Chl gFw-1)
Acetylene reduction activity (nmol h -1 nodule-1)
S. Chungopast et al. / Journal of Plant Physiology 171 (2014) 104–108
B
nitrogen-fixing activity of the nodules (Pladys et al., 1991). When we measured leghemoglobin gene expression in the nodules, the STM30-inoculated nodules had reduced expression compared with the WT-inoculated nodules (Fig. 4B). These data strongly support the hypothesis that STM30 inoculation induces early nodule senescence. Characteristics of the plants inoculated with STM30 or WT M. loti When the nodules inoculated with STM30 became greener, the leaf color appeared yellowish (Fig. 5). When we measured the chlorophyll content in the leaves, the values for the plants inoculated with STM30 was 35% lower than the leaves from the WT-inoculated plants at 7 wpi. These data suggest that the GSI in the bacteroids may be detrimental to the symbiosis. Nitrogen-fixing plants can be classified as amide or ureide exporters based on the composition of the xylem fluid collected from the excised nodules (Schubert, 1986). The temperate legume L. japonicus exports asparagine or glutamine out of the nodule to the shoot as the major products of N2 fixation (Schubert, 1986). We measured the free amino acid content of glutamine, asparagine, glutamate and aspartate in the bacteroids, nodules and leaves at 7 wpi (Table 1). Remarkably, the concentration of free amino acids, especially glutamate, glutamine and asparagine, in the bacteroids, nodules and leaves inoculated with STM30 were lower compared with the plants inoculated with the WT M. loti. These data suggest that the legume–rhizobia symbiosis is severely affected by STM30.
30
Discussion
25 20
**
15
*
10 5 0 4
7 Weeks post inoculation
Fig. 4. Expression patterns of the cysteine protease (A) and leghemoglobin (B) genes in the nodules inoculated with STM30 () or wild-type ( ) M. loti. The qRT-PCR analysis was performed using the cDNA from the nodules harvested at four and seven weeks post-inoculation. The expression was normalized to ubiquitin. All of the data are shown as ±SD. Statistically significant differences compared with the wild-type nodules are indicated with * (P < 0.05) or ** (P < 0.01).
STM30 is a mutant strain containing a Tn insertion in the GSI gene (Fig. 1A). Although there are nine GS family genes in the M. loti genome, there is only one GSI gene (mll0343). When we produced other GS mutants by inserting a Tn in each gene (mlr0339, mlr6210, mll7254, mll7307, mll5148, and mll6521), we could not detect any nodule senescence, except for the STM30 mutant (data not shown). The nodule number per plant infected with STM30 increased continuously throughout the growth period (Fig. 2A). Many of the nodules for the plants infected with STM30 carried senesced green nodules (Fig. 2B). The ARA per STM30-inoculated nodule was significantly lower compared with the nodules inoculated with WT M. loti (Fig. 3A). As a biochemical indicator of nodule senescence, cysteine protease activity for degrading leghemoglobin has been reported
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Table 1 The free amino acids in bacteroids and leaves of L. japonicus inoculated with wild-type or STM30 mutant. Values are the mean ± standard deviations (n = 5). Statistical significant differences compared with wild-type are indicated with * (P < 0.05) and ** (P < 0.01). Free amino acid
Nodules
Bacteroids
Wild-type −1
(nmol gFw Glu Gln Asn Asp
STM30
Wild-type
Leaves STM30
Wild-type
STM30
) 602.37 39.22 2350.87 61.55
± ± ± ±
105.75 8.66 262.16 15.53
274.52 21.98 1315.73 51.84
± ± ± ±
125.02* 6.91* 186.75** 21.16
26.67 1.36 41.11 3.55
(Pladys and Rigaud, 1985; Pladys et al., 1991). Early nodule senescence has shown to lead to a higher expression of cysteine protease genes (Pladys and Vance, 1993). After inoculation with STM30, many green nodules, apparently lacking intact leghemoglobin, appeared; in addition, cysteine protease expression increased (Fig. 4A), whereas leghemoglobin expression decreased (Fig. 4B). B. japonicum glnA or glnB single mutants have been reported to produce an increased number of nodules (Carlson et al., 1987). However, the reason for this increase in nodule number was not clear. We speculate that the B. japonicum glnA single mutant might also induce early nodule senescence. Taken together, these data suggest that the nodules inoculated with STM30 had reduced nitrogen-fixing activity per nodule and early nodule senescence. To maintain the nitrogen fixing activity, the plants inoculated with STM30 might produce many nodules. Early nodule senescence also induces plant senescence. The chlorophyll concentration in the leaves of STM30-infected plants was lower compared with the leaves of the WT infected plants. Temperate legumes, such as L. japonicus, pea, clover, and alfalfa, mainly export glutamine or asparagine out of the nodules to the shoot as a nitrogen compound (Temple et al., 1998; Goggin et al., 2003). The concentration of free amino acids, such as glutamine and asparagine, in the leaves of STM30-infected plants was lower (Table 1). The bacteroids with GSI deficiency did not fix nitrogen effectively and might not produce enough nitrogen compounds, such as glutamine and asparagine, in the nodules (Fig. 3 and Table 1). The lower chlorophyll concentration in the plants inoculated with STM30 might be the reason that glutamine and asparagine could not be effectively exported to the shoot. In recent years, one of the two amino acid uptake systems (Aap and Bra) in Rhizobium leguminosarum has been reported to be essential for symbiotic nitrogen fixation in pea nodules (Lodwig et al., 2003). In the absence of an amino acid transport system between the bacteroids and the plant cytoplasm, the bacteroids cannot efficiently fix nitrogen. Another report has indicated that an increase in the intracellular glutamine/glutamate ratio triggers the post-transcriptional downregulation of amino acid uptake by Aap and Bra (Mulley et al., 2011). The glutamine/glutamate ratio in bacteroids inoculated with STM30 was two-fold higher compared with nodules with the WT M. loti (Table 1), suggesting that STM30 also might suppress amino acid transport via Aap/Bra. Thus, we conclude from these data that the GSI in bacteroids plays an important role in maintaining legume–rhizobia symbiosis. A biochemical survey of the role of GSI in bacteroids for protecting nodules from senescence is underway in our laboratory. Acknowledgments This study was supported by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank the National BioResource Project for the Mesorhizobium loti STM30 mutant (STM clone ID, 10T05g06). C.S. was supported by Thailand government scholarship.
± ± ± ±
0.41 0.16 8.01 0.86
8.47 0.95 25.67 3.80
± ± ± ±
1.03** 0.08** 3.22** 0.34
1198.70 76.31 1239.90 296.52
± ± ± ±
112.24 2.92 70.53 15.10
944.05 41.51 592.61 283.95
± ± ± ±
72.73** 8.09** 24.19** 3.28
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