Advance Access published May 28, 2003 Journal of Experimental Botany, Page 1 of 6 DOI: 10.1093/jxb/erg189
RESEARCH PAPER
Nitrate-independent expression of plant nitrate reductase in Lotus japonicus root nodules Kazuhisa Kato, Yoshimichi Okamura, Koki Kanahama and Yoshinori Kanayama* Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan Received 9 January 2003; Accepted 3 April 2003
Nitrate-independent nitrate reductase (NR) activity is generally found in legume root nodules. Therefore, the effects of nitrate on plant NR activity and mRNA were investigated in the root nodules of Lotus japonicus (L. japonicus). Both NR activity and mRNA levels in roots and root nodules were up-regulated by the addition of nitrate. In the absence of nitrate, NR activity and mRNA were detected in root nodules but not in roots. Southern blotting analysis indicates that NR is encoded by a single gene in L. japonicus. No nitrate was detected in the root nodules or roots of plants grown in the absence of nitrate, while its accumulation was observed in plants supplied with exogenous nitrate. These results indicate that inducible-type NR can be expressed in root nodules in the absence of nitrate. The activation state of the nitrate-independent activity of NR was as high as that of NR activity induced by nitrate. NR mRNA expressed independently of nitrate in root nodules without nitrate was localized in the infected regions of the root nodules. Thus, the expression could be related to the speci®c structure and environment of root nodules. Key words: Lotus japonicus, nitrate, nitrate reductase, nitrogen ®xation, root nodule.
Introduction Legumes with root nodules can utilize both nitrogen gas from the atmosphere and inorganic nitrogen in the soil. Nitrate is ®rst reduced to nitrite by nitrate reductase (NR), while gaseous nitrogen is ®xed by nitrogenase. However,
root nodules do not use nitrogen gas ef®ciently in the presence of a signi®cant level of nitrate in the soil, because nitrate inhibits nodulation and nitrogenase activity (Streeter, 1985a, b). Although the mechanism of the inhibitory effect of nitrate on nitrogen ®xation is not yet clear, several researchers have suggested a role for nitrate reduction in the inhibitory effect. Arrese-Igor et al. (1997, 1998) reported that in soybean root nodules nitrate gained access to an infected region in the short-term, and was reduced to nitrite, leading to the formation of nitrosylleghaemoglobin (LbNO). Plant NR has been shown to produce nitric oxide (NO) from nitrite using NADH (Yamasaki et al., 1999; Yamasaki and Sakihama, 2000). As leghaemoglobin plays a key role in oxygen supply to bacteroids in an infected region, NRmediated formation of LbNO could affect nitrogenase activity directly or indirectly (Kanayama and Yamamoto, 1990; Vessey and Waterer, 1992; Arrese-Igor et al., 1998). In pea, the decrease in nitrogenase activity caused by nitrate was less in NR-de®cient mutants than in wild-type plants (Walsh and Carroll, 1992; Kaiser et al., 1997), suggesting a relationship between NR and decreased nitrogenase activity. Takahashi et al. (1992) showed that nitrate metabolism in root nodules, rather than that in roots, is important in the nitrate-induced inhibition of nitrogenase activity. Although NR is present in both bacteroids and the cytosol (plant fraction) in root nodules, bacteroid NR is not involved in the nitrate-induced inhibition of nitrogenase activity (Streeter, 1985b). Thus, the focus here was on plant NR in the nodule cytosol. NR activity is generally induced by nitrate, although some species possess constitutive types of NR. Ammonium ions and phytohormones may induce NR activity without nitrate in special cases (Solomonson and
* To whom correspondence should be addressed. Fax: +81 (0)22 717 8642. E-mail:
[email protected] Abbreviations: L. japonicus, Lotus japonicus; M. loti, Mesorhizobium loti; NR, nitrate reductase. Journal of Experimental Botany, ã Society for Experimental Biology 2003; all rights reserved
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Abstract
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Materials and methods Plant material L. japonicus cv. Gifu was sown in H2O/agar medium (Takara, Kyoto, Japan) and grown at 25 °C under an 18 h photoperiod. After 2 weeks, plants were inoculated with Mesorhizobium loti (M. loti) (strain nzp2235) and repotted in vermiculite. The plants were supplied with a nitrogen-free nutrient solution (Matsumoto et al., 1977) until approximately 52 d after sowing and then supplied with 10 mM KNO3 or 10 mM KCl (Control) for 5 d. Roots and root nodules were stored for the experiments at ±80 °C. Molecular cloning of NR cDNA fragment from root nodules Three degenerated primers were designed on the basis of three conserved regions of NR from 12 higher plant NRs (Hoff et al., 1992) for RT-PCR using RNA PCR Kit Version 2.1 (Takara). Their nucleotide sequences are 5¢-RAAYTCNGTRCARTC-3¢ for primer 1, 5¢-TNTGYGCNGTRCARTC-3¢ for primer 2, and 5¢-TNTGYGCNGTRCARTC-3¢ for primer 3 (N, A+T+G+C; M, A+C; R, A+G; Y, C+T). Total RNA was extracted from root nodules supplied with nitrate using RNeasy MiniKit (Qiagen, Chatsworth, USA). The ®rst strand cDNA was synthesized from 1 mg of total RNA by using primer 1. Subsequent PCR reaction was achieved through 40 and 27 cycles for ®rst and second PCR, respectively, of 94 °C for 30 s, 40 °C for 30 s, and 72 °C for 1 min. The ®rst PCR with primer 1 and primer 2 gave a predicted fragment of 921 bp. This fragment was subjected to the second PCR with primer 1 and primer 3 and an 870 bp fragment was ampli®ed. The product that was cloned into a pT7Blue vector (Novagen, Madison, USA) proved to be part of an NR cDNA by sequencing and comparison to amino acid sequences of other plant NR. Nucleotide sequencing was carried out using the 373A DNA Sequencing System (Applied Biosystems, Foster City, USA). Southern and northern analyses For Southern analysis, DNA extraction from plants, electrophoresis, and blotting were carried out according to Kanayama et al. (1997). Genomic DNA from M. loti was extracted by the method of Wilson (1995). The blotted membrane was hybridized against a digoxigenin (DIG) DNA probe prepared from the PCR fragment of NR using a PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany). The ®lter was then washed at 65 °C in 0.23 SSC/0.1% (w/v) SDS and exposed to Fuji Medical X-ray ®lm (Fuji Film, Tokyo, Japan). Total RNA (2 mg) was extracted using the RNeasy Minikit (Qiagen) from each sample, separated on a 3-(N-morpholino)propanesulphonic acid/1.2% (w/v) agarose/5% (w/v) formaldehyde denaturing gel, and transferred to a Hybond N+ membrane. The
membrane was hybridized with a DIG-labelled RNA probe that was prepared from the linearized plasmids harbouring the PCR fragment of NR by using DIG RNA labelling kit (Roche Diagnostics). The hybridization and washing procedures followed the manufacturer's instruction. Nitrate content, NR activity, and activation state of NR Nitrate content was assayed by the method of Cataldo et al. (1975). In vitro NADH-NR activity and its activation state were assayed according to Foyer et al. (1998) except that 200 mM phenylmethylsulfonyl¯uoride (PMSF) and 1% (w/v) bovine serum albumin (BSA) were added to the extraction buffer. In situ hybridization In situ hybridization was carried out according to modi®cations of the method of Kouchi and Hata (1993) and Kanayama et al. (1998). Root nodules that were not supplied with nitrate were ®xed with 4% (w/v) paraformaldehyde and 0.25% (v/v) glutalaraldehyde in 10 mM sodium phosphate buffer (pH 7.4) overnight at 4 °C. They were then dehydrated in a graded ethanol series, substituted with xylene, and embedded in histoparaf®n (Wako, Osaka, Japan). Microtome sections (7 mm thick) were applied to silane-coated glass slides (Dako, Kyoto, Japan). Tissues were deparaf®nized with xylene, and rehydrated through a graded series of ethanol. They were incubated with 0.2 N HCl for 20 min, and then with 5 mg ml±1 proteinase K in 100 mM TRIS-HCl (pH 7.5) containing 50 mM EDTA for 30 min at 37 °C to digest protein. The sections were treated with 0.1 M triethanolamine (pH 8.0) for 5 min, and then with 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min to acetylate any remaining positive charge, and then incubated with 23 SSC. The hybridization solution contained 50% (v/v) formamide, 300 mM NaCl, 10 mM TRIS-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 13 Denhardt's solution, 125 mg ml±1 herring sperm DNA, 125 mg ml±1 yeast tRNA, 0.25% (w/v) SDS, 10% (w/v) dextran sulphate, and DIG-labelled RNA probes prepared for northern analysis at the concentration of 1 mg ml±1. RNA probes were restrictively hydrolysed at approximately 100 base. Hybridization was carried out at 50 °C for 16 h. Then the slides were washed four times in 43 SSC at 50 °C for 10 min. The excess RNA probes were removed by incubation at 37 °C for 30 min in a solution containing 10 mM TRISHCl (pH 7.5), 500 mM NaCl, 5 mM EDTA, and 10 mg ml±1 RNase A. The slides were then washed twice with the same solution without RNase A for 10 min at 37 °C, 23 SSC for 30 min at 55 °C, and 0.13 SSC for 30 min at 55 °C. The hybridization signals were detected by antidigoxigenin-alkaline phosphatase (Roche Diagnostics). After colour development with 4-nitroblue tetrazolium chloride and 5bromochloro-3-indolylphosphate, the slides were washed in distilled water and mounted in Permount (Fisher Scienti®c, Fair Lawn, USA). In this experiment, DIG-labelled sense RNA probes of NR cDNA were used as a control.
Results and discussion Cloning and Southern blotting analysis
Root nodule NR was cloned by RT-PCR from L. japonicus supplied with nitrate. The nucleotide sequence of the PCR fragment (834 bp) was the same as that of the exon alignment of the L. japonicus NR genomic clone (accession number X80670). No PCR fragments with different sequences were obtained, although degenerate primers based on the conserved sequence of plant NR (Hoff et al., 1992) were used for RT-PCR. The amino acid sequence deduced from the PCR fragment showed 90% and 85%
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Barber, 1990; Bungard et al., 1999). However, ammonium ions or amino acids usually have little effect or an inhibitory effect on NR activity, and NR is a typical substrate-induced enzyme. Interestingly, nitrate-independent NR activity has been observed in the root nodules of several legumes (Arrese-Igor et al., 1990; Caba et al., 1995; Kanayama et al., 1999; Silveira et al., 2001). Kanayama et al. (1999) suggested that in the soybean, the nitrate-independent activity was due to the inducible type of plant NR. Despite this unique characteristic, plant NR in root nodules has not been studied at the molecular level. To determine the function of nodule NR, a molecular analysis of NR was performed using L. japonicus, a model legume that is amenable to genetic manipulation.
Nitrate reductase in root nodules 3 of 6
the intron of the NR genomic clone contained an XbaI site at nucleotide position 248 of the probe, two bands were detected on digestion with this endonuclease. By contrast, no band was detected using DNA from M. loti. These results indicate that the NR cloned from L. japonicus root nodules was a single gene and that the probe did not crosshybridize with bacterial genes. The complete nucleotide sequence of the M. loti genome has been reported (Kaneko et al., 2000). The nucleotide sequence of NR (NP_104103) in the M. loti genome has a very low level of homology (39% identity) to that of L. japonicus NR.
Fig. 2. Genomic Southern analysis of L. japonicus NR gene. Genomic DNA (5 mg) from L. japonicus and M. loti was digested with EcoRI (E), XbaI (X) and HindIII (H).
identity to the sequences of NR of Phaseolus vulgaris (U01029) and Nicotiana tabacum (X14059), respectively (Fig. 1). Southern hybridization was carried out using the PCR fragment as a probe. In L. japonicus, a single band was detected in both EcoRI and HindIII digests (Fig. 2). As
NR mRNA expression and activity, and nitrate contents in roots and root nodules On northern analysis, NR mRNA was not detected in roots without nitrate, and was induced by the addition of nitrate (Fig. 3). The NR activity in the roots corresponded to the mRNA level (Table 1). These results indicate that root NR in L. japonicus is of the typical nitrate-inducible type. On the other hand, NR mRNA was detected in root nodules, even without nitrate, and its level increased following addition of nitrate (Fig. 3). This change in the mRNA level was also re¯ected in the NR activity (Table 1). The nitrate contents of root nodules and roots were measured to investigate the in¯uence of nitrate accumu-
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Fig. 1. Alignment of deduced amino acid sequence of L. japonicus NR cDNA cloned from root nodules with Phaseolus vulgaris and Nicotiana tabacum NR. Accession numbers are U01029 (Phaseolus vulgaris PVNR2) and X14059 (Nicotiana tabacum nia-2).
4 of 6 Kato et al. Table 1. NR activity (mmol g±1FW h±1) in response to the addition of 10 mM nitrate for 5 d NR activation state is de®ned as the ratio of activity assayed in the presence of Mg2+ (NRAact) to activity in the presence of EDTA (NRAmax). Nodule
Root
Control NRAmax NRAact NR activation state (%) a
a
0.07660.004 0.05060.002 65.9
Nitrate
Control
Nitrate
0.32560.042 0.20360.039 62.3
Trace Trace ±
0.29160.013 0.13560.008 46.6
Mean 6 standard error (n=3).
Fig. 4. Nitrate content in L. japonicus root nodules (N) and roots (R) were supplied with (Nitrate) or without (Control) nitrate for 5 d. Vertical bars indicate standard errors (n=3). ND means not detected.
lation on the expression of NR. Nitrate was not detected in the root nodules or roots of plants grown in the absence of nitrate, while its accumulation was observed following addition of nitrate (Fig. 4). These observations indicate that nitrate accumulation is not required for gene expression of NR in root nodules, although the accumulation of nitrate enhances its expression. Activation state of NR
Plant NR is inactivated by phosphorylation and the binding of 14-3-3 proteins (Campbell, 1999; Kaiser and Huber,
2001; MacKintosh and Meek, 2001). The activation state of NR in leaves has been investigated in detail with regard to the effects of light (Kaiser and Huber, 2001). The present study estimated the activation state in root nodules for the ®rst time, and showed it to be similar in root nodules with and without nitrate supply (Table 1). In addition, the percentage of active NR was slightly higher in root nodules than in roots. These results suggest that root-nodule NR expressed independently of nitrate is functionally similar to NR induced by nitrate in roots and root nodules. Spatial expression pattern of the NR gene Nitrate-independent expression of the NR gene was observed in root nodules, despite the absence of NR mRNA in the roots. Therefore, expression could be related to the nodule-speci®c structure. Analysis by in situ hybridization indicated that the transcripts in root nodules without nitrate were localized within the infected region and the vascular tissue (Fig. 5). No signi®cant signals were observed using the sense probe. It is possible that the localization of NR mRNA is related to environmental factors that are speci®c to the infected regions of root nodules, such as low free oxygen concentration. This hypothesis is supported by the high NR activity reported under conditions of anoxia (Botrel and Kaiser, 1997).
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Fig. 3. Northern blot analysis of L. japonicus NR. Root nodules (N) and roots (R) were supplied with (Nitrate) or without (Control) nitrate for 5 d. Total RNA (2 mg) from each sample was subjected to the analysis. rRNA stained with ethidium bromide is shown as loading control.
Fig. 5. In situ localization of NR mRNA in L. japonicus root nodules. Nodules were harvested from plants in the absence of nitrate. Transverse sections (7 mm) through the nodules were hybridized with DIG-labelled antisense (A) and sense (B) RNA probes. Hybridization signals are visible as blue. The bar represents 250 mm.
Nitrate reductase in root nodules 5 of 6
Conclusions
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