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First published online April 27, 2007; 10.1104/pp.107.097063 Plant Physiology 144:806-820 (2007) © 2007 American Society of Plant Biologists
TILLING Mutants of Lotus japonicus Reveal That Nitrogen Assimilation and Fixation Can Occur in the Absence of Nodule-Enhanced Sucrose Synthase[C],[W]Metabolic Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom (I.H., T.W., T.L.W.); Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom (S.K.); Kazusa DNA Research Institute, Kisarazu, Chiba 292–0818, Japan (T.K., S.S., S.T.); and Department of Biology 1, Ludwig-Maximilian-Universität, D–80638 Munich, Germany (M.P.)
In all plant species studied to date, sucrose synthase occurs as multiple isoforms. The specific functions of the different isoforms are for the most part not clear. Six isoforms of sucrose synthase have been identified in the model legume Lotus japonicus, the same number as in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). The genes encoding these isoforms are differentially expressed in all plant organs examined, although one, LjSUS4, is only expressed in flowers. LjSUS1 is the most highly expressed in all plant organs tested, except root nodules, where LjSUS3 accounts for more than 60% of the total SUS transcripts. One gene, LjSUS2, produces two transcripts due to alternative splicing, a feature not observed in other species to date. We have isolated plants carrying ethyl methanesulfonate-induced mutations in several SUS genes by targeting-induced local lesions in genomes reverse genetics and examined the effect of null alleles of two genes, LjSUS1 and LjSUS3, on nodule function. No differences were observed between the mutants and wild-type plants under glasshouse conditions, but there was evidence for a nitrogen-starvation phenotype in the sus3-1 mutant and severe impairment of growth in the sus1-1/sus3-1 double mutant under specific environmental conditions. Nodules of sus3-1 mutant plants retained a capacity for nitrogen fixation under all conditions. Thus, nitrogen fixation can occur in L. japonicus nodules even in the absence of LjSUS3 (the major nodule-induced isoform of SUS), so LjSUS1 must also contribute to the maintenance of nitrogen assimilation.
Nitrogen fixation in bacteroids and conversion of ammonia to amino acids in the infected cells of legume nodules require energy (ATP), carbon skeletons, and reductants that are produced from Suc synthesized in the leaves and imported into the nodules (Vance and Heichel, 1991  ; Gordon, 1995; Udvardi and Day, 1997). Two types of enzymes could in theory be responsible for the initial cleavage of imported Suc in nodule cells: Suc synthase (EC 2.4.1.13) and invertase (EC 3.2.1.26). Suc synthase cleaves Suc into Fru and UDP-Glc and is reversible in vivo (Geigenberger and Stitt, 1993). In contrast, cleavage by invertases leads to the formation of Fru and Glc and is irreversible (Avigad, 1982). In general, Suc synthase is believed to be the main route of Suc catabolism in organs in which oxygen tension is low (Guglielminetti et al., 1995; Rolletschek et al., 2002) because conversion of Suc to hexose phosphates via Suc synthase requires less ATP than conversion via invertase. Suc synthase activity is also important for the provision of energy for phloem loading (Martin et al., 1993) and in the development of sink strength (Edwards and ap Rees, 1986), two other processes important in nodulation.
Suc synthase is encoded by a small multigene family in all species analyzed to date, including pea (Pisum sativum; Barratt et al., 2001
Vacuolar and cell wall acid invertases have essential roles in cell expansion (Sergeeva et al., 2006
We have chosen the model legume L. japonicus to dissect genetically the transit of carbon through nodules and to develop an understanding of the metabolic factors that influence the rate of nitrogen fixation in nodules. In this article, we report the identification of six genes encoding Suc synthase in L. japonicus and their patterns of expression in the plant. We used our recently developed TILLING (for targeting-induced local lesions in genomes) platform (Perry et al., 2003
There Are Six Isoforms of Suc Synthase in L. japonicus
There are at least six genes encoding different isoforms of Suc synthase in L. japonicus (Fig. 1
), which have been found by exhaustive analysis of the published genome or sequences that are available at the Kazusa DNA Research Institute. The first three genes were identified via assembly of EST sequences followed by screening of the public databases or transformation-competent artificial chromosome (TAC)/bacterial artificial chromosome libraries for corresponding genomic sequences as described in "Materials and Methods." LjSUS4 was identified by analyzing TAC clones for sequence similarities to LjSUS1 and LjSUS3. LjSUS5 and LjSUS6 were found by comparison with known Arabidopsis sequences for Suc synthases 5 and 6 (Baud et al., 2004
LjSUS2 differed from the genes encoding other family members with regard to a very long intron at its 3' end. Analysis of EST clones revealed that LjSUS2 can give rise to two different mRNAs, referred to as LjSUS2 and LjSUS2a (Fig. 2 ). In LjSUS2, the last intron, with a length of 5.4 kb, is spliced. The last exon of LjSUS2 consists of 21 nucleotides, coding for six amino acids and a stop codon, and the 3'-untranslated region (UTR). In LjSUS2a, the last intron is retained in the mRNA. This alternative splicing transforms the intron of LjSUS2 to an exon consisting of six nucleotides, coding for one amino acid and a stop codon, and the 3' UTR. The two mRNAs are predicted to give rise to two different proteins, LjSUS2 being five amino acids longer than LjSUS2a. The phylogenetic relationship between the L. japonicus Suc synthase isoforms and those of other species was constructed from their deduced amino acid sequences (Fig. 3 ). It showed clearly that the three pairs of L. japonicus isoforms fit into the three respective classes of isoforms as noted elsewhere (Komatsu et al., 2002 ).
Suc Synthase Genes Are Differentially Expressed in L. japonicus Quantitative reverse transcription (qRT)-PCR experiments were performed (Table I) to analyze the expression patterns of the different Suc synthase genes in L. japonicus. All Suc synthase genes, except LjSUS4, were expressed in all organs analyzed (Fig. 4, A–H ). Calculations of the contribution of each gene to overall Suc synthase transcript levels have been made based on the assumption that L. japonicus has six isoforms of Suc synthase. LjSUS1 was the gene most highly expressed in the majority of organs examined, contributing more than 60% to the total amount of Suc synthase transcript (Fig. 4, A–E, and H). LjSUS2 and LjSUS2a were expressed at levels between 2% and 8% in all organs examined, except leaves, where they contributed up to 30% of total Suc synthase transcript (Fig. 4, A and B). LjSUS2 was more abundant than LjSUS2a in all organs analyzed. LjSUS3 transcript contributed between 9% and 29% of transcript in most organs (Fig. 4, A–E, and H), but more than 60% of the total Suc synthase transcript in inoculated roots and nodules (Fig. 4, F and G). LjSUS4 was detected only in flowers, where it contributed less than 1% of total Suc synthase transcript (Fig. 4H). LjSUS5 and LjSUS6 were expressed in all organs, but at very low levels. Only in young leaves were they expressed to an extent greater than 1% (Fig. 4A). When expression of the different genes was compared between plant organs relative to their expression in young leaves (Fig. 5 ), it became apparent that expression of LjSUS3 is enhanced in nodules and inoculated roots (Fig. 5D). A similar enhancement was not found for any other gene in any other organ (Fig. 5, A–C, and E and F). Hence, LjSUS3 can be regarded as a nodule-enhanced SUS gene.
Mutants Generated by TILLING Reverse Genetics Are Impaired in Suc Synthase Activity Several nonsilent mutations for LjSUS1 to LjSUS4 were identified via TILLING: 17 for LjSUS1, six for LjSUS2, four for LjSUS3, and three for LjSUS4 (Fig. 6, A–D ). The large number of mutants in the LjSUS1 gene was found because the whole genomic sequence was analyzed using overlapping PCR fragments. This was done as part of a general exercise to examine the depth of allelic variation in the ethyl methanesulfonate-mutagenized population (see Supplemental Tables S1–S4 for full details). For the other genes, only part of the genomic sequence was analyzed. The mutants predicted by CODDLE (for codons optimized to discover deleterious lesions) to have potentially deleterious effects on gene function were subjected to further analysis: five alleles for LjSUS1, three for LjSUS3, and two for LjSUS2 and LjSUS4 (Fig. 6, A–D). One allele for each of LjSUS1 and LjSUS3 was found to have a premature stop codon in the middle of the sequence: at amino acid 384 for the line isolated from SL649-1 (sus1-1) and at amino acid 376 for the line isolated from SL3154-1 (sus3-1). Due to the high transcript levels of the genes encoding both of these isoforms in all organs, including nodules (Fig. 4, A–H), we focused on sus1 and sus3 mutants to elucidate the role of these Suc synthase isoforms in nodule metabolism. We also generated a double mutant, sus1-1/sus3-1.
Nodules of sus1-1 had 15% of wild-type levels of LjSUS1 transcript, and nodules of sus3-1 had 23% of wild-type levels of LjSUS3 transcript (Fig. 7 ). Hence, the aberrant stop codons in sus1-1 and sus3-1 probably resulted in nonsense-mediated decay of the aberrant LjSUS1 and LjSUS3 transcripts, respectively. Interestingly, qRT-PCR data also showed that sus1-1 mutants had 7-fold increased LjSUS2 and LjSUS2a levels, whereas they were unchanged in sus3-1 mutants (Fig. 7).
Suc synthase activity was measured in nodule extracts of sus1 and sus3 homozygous mutants predicted to carry a deleterious mutation and in wild-type plants. A statistically significant difference in the activity between mutant and wild-type plants was observed for all sus3 mutants analyzed, but not for any of the sus1 mutants (Table II ). Lines sus1-1 and sus3-1 were then backcrossed a second time (see "Materials and Methods"), and the SUS activity in nodules was compared to that in their respective wild-type plants. The sus1-1 mutant showed a 38% reduction, and the sus3-1 mutant showed a 67% reduction in Suc synthase activity compared to their respective wild-type levels (significant decrease at the 5% level; Student's t test). The activity of Suc synthase in the leaves was also compared between sus1-1 and wild type because LjSUS1 transcript made a greater contribution to leaf Suc synthase transcript (Fig. 4, A and B) than to nodule Suc synthase transcript (Fig. 4G). Activity in sus1-1 leaves was about 50% of the activity measured for the wild type (0.145 ± 0.005 compared to 0.294 ± 0.021 µmol min–1 mg–1 protein; means ± SE from three individual plants). It seems likely from these results that sus1-1 and sus3-1 alleles give rise to little or no functional corresponding SUS protein.
Further evidence to support the idea that the sus3-1 allele does not give rise to protein came from western-blot analysis of nodule proteins. An antiserum against PsSUS3 was used that detects all three known isoforms in pea (Barratt et al., 2001 ) and all isoforms in Arabidopsis, except AtSUS5 (P. Barratt, personal communication). Western blots showed that sus3-1 plants had decreased Suc synthase content in nodules (Fig. 8
). No difference was observed in the content of Suc synthase protein between nodules of wild-type and sus1-1 plants; any difference in SUS1 protein content was likely to be small. This was supported by the fact that the expression of LjSUS1 in nodules is relatively low compared with LjSUS3 (Fig. 4G). The relative transcript levels and the measurements of enzyme activity (Table II) indicate that LjSUS3 makes a larger contribution to both SUS transcript and Suc synthase activity in nodules than LjSUS1.
Primary metabolites in nodules at 57 d postinoculation (dpi) from plants grown in a mixture of perlite and vermiculite were analyzed by gas chromatography (GC)-mass spectrometry (MS). The analysis showed that sus3-1 mutants had significantly greater Suc content (Student's t test at the 5% level) than wild-type nodules (7.13 ± 0.22 and 2.20 ± 0.02 µmol g–1 dry weight, respectively; means ± SE from three extractions). Furthermore, we confirmed that alkaline invertase activity was not up-regulated in both sus1-1 or sus3-1 mutants, and acid invertase activity was not measurable (data not shown). These data are consistent with the idea that the sus3-1 mutant has severely reduced capacity for Suc metabolism.
Suc synthase mutants were grown in several different media in a glasshouse. A medium that gives excellent root and nodule growth is Terragreen (a calcined attapulgite clay) mixed 1:1 with sand. This is frequently used for arbuscular mycorrhiza experiments. In this medium, as well as in compost, sus1-1 and sus3-1 mutants were not visibly different from wild-type plants. Although Terragreen has been reported to be inert in some publications (Boddington and Dodd, 1999
Growth parameters of wild-type plants, sus1-1, and sus3-1 mutants in the CER were recorded over a time course. Plants were inoculated with rhizobia 5 d after germination and watered twice weekly with nitrogen-free nutrient solution. Control plants were watered with Hornum solution containing nitrogen (see "Materials and Methods"). Measurements of height, the number of branches, expanded leaves, flowers, and pods, the fresh and dry weight above and below ground revealed no differences between the mutant and corresponding wild-type plants before 34 dpi (data not shown). At 47 dpi, sus1-1 mutants were the same as the corresponding wild-type plants in all parameters measured, whereas sus3-1 plants started to differ from wild-type plants in various aspects (Table III ): The number of fully expanded leaves and the number of branches of sus3-1 plants were decreased and basal leaves were yellow (Fig. 9A). There was no difference between the plants in height (Table III), the numbers of flowers, or the time of flower and pod formation (data not shown). The phenotype of sus3-1 plants became more pronounced as the plants aged and an extreme example is shown in Figure 9A. The lower leaves of sus3-1 mutants had a much lower chlorophyll a/b content than those of wild-type plants, whereas sus1-1 and wild-type plants did not differ from each other in this respect (Table IV ). Seed pods were harvested as they matured and seed fresh and dry weights were determined. There were no significant differences between sus1-1 and wild-type plants, whereas both the fresh and dry weight of seed was decreased in sus3-1 plants compared to wild type (Table IV). sus3-1 mutants did not show yellowing or growth retardation relative to wild-type plants when grown with the addition of nitrogen fertilizer (data not shown). These results indicated that sus3-1 plants suffered from nitrogen starvation in the absence of added nitrogen.
The sus3-1 Mutant Has Functional Nodules
An acetylene reduction assay was used to analyze the capacity of nodules for nitrogen fixation. At 57 dpi, nodules of wild type, sus1-1, and sus3-1 plants grown without nitrogen fertilizer in the CER were healthy and pink and showed the same amount of acetylene reduction, regardless of whether ethylene production per nodule or ethylene production per root system was determined (Table V
). There was also no difference between wild-type and mutant plants in nodule number or the number of senescent nodules (Table V). To analyze the nitrogen status of mutant plants,
Growth of the sus3-1/sus1-1 Double Mutant Is Severely Impaired in the Absence of Nitrogen Plants homozygous for the sus3-1 allele may be able to rely on LjSUS1 to provide sufficient carbon for fixation because they were able to grow normally under certain circumstances. We generated a double mutant, sus1-1/sus3-1, therefore, to shed further light on the importance of LjSUS1 and LjSUS3 in Suc metabolism in the nodule. Nodules of the double mutant showed a much more severe reduction in Suc synthase activity relative to wild-type plants than did those of sus3-1 mutants. Nodules from double mutants grown in a CER without added nitrogen had 94% lower activity than wild-type plants grown in the same condition (0.28 ± 0.06 compared with 4.63 ± 0.13 µmol min–1 g–1, means ± SE for three individual plants), and Suc synthase protein was undetectable on western blots (Fig. 8). Under these growth conditions, double mutants started to differ visibly from the corresponding wild-type plants earlier than sus3-1 mutants, at 38 dpi. Leaf number was much more strongly reduced than that of sus3-1 mutants, and height was also reduced, whereas it was not affected in sus3-1 mutants (data not shown; Table III). At 47 dpi, double mutants had yellow leaves and at 57 dpi they had a significantly reduced shoot weight, but did not differ in root weight from wild-type plants (double-mutant shoot weight was 0.73 ± 0.14 g and wild-type weight was 1.54 ± 0.22 g; double-mutant root weight was 0.61 ± 0.21 g and wild-type weight was 0.86 ± 0.15 g; all values are means ± SE of three replicates). When plants were fed with nitrogen-containing nutrient solution, their appearance was very similar to the wild type (Fig. 9B) and there were no differences with respect to chlorophyll content or shoot and root weight (data not shown).
We have shown that Suc synthase is encoded by a small family of at least six genes in L. japonicus (Fig. 1). This is the same number of genes reported for Arabidopsis (Baud et al., 2004 ) and rice (Huang et al., 1996; Harada et al., 2005) and extends the understanding of the gene family in legumes, where several isoforms have been reported in Medicago truncatula (Hohnjec et al., 1999) and pea (Barratt et al., 2001). As in Arabidopsis, the encoded SUS proteins appear to fall into three distinct groups of isoforms, each group containing two proteins (Fig. 3). In L. japonicus, these pairs of SUS proteins are LjSUS1 and LjSUS3, LjSUS2 and LjSUS4, and LjSUS5 and LjSUS6. The equivalent protein pairs in Arabidopsis are AtSUS1 and AtSUS4, AtSUS2 and AtSUS3, and AtSUS5 and AtSUS6, respectively. The similarities between the amino acid sequences of the isoforms belonging to the different groups between species suggest that the genes encoding members of the different groups diverged a relatively long time ago, at least before the separation of monocots and dicots. Conservation of sequence differences over such long periods of time usually reflects functional differences between members of the different subclades. The divergence between the genes encoding the pairs of proteins belonging to each subclade appears to have been more recent. Interestingly, LjSUS3, the gene most highly expressed in nodules of L. japonicus, is closely related to pea SUS1 (PsSUS1), which is known to have an important function in carbon metabolism in nodules (Craig et al., 1999). Unlike other species studied so far, a subisoform of LjSUS2, called LjSUS2a, was predicted as a result of alternative splicing of the LjSUS2 transcript (Fig. 2). This leads to the formation of two different mRNAs. LjSUS2a was present at very low levels in all organs analyzed and was always lower than LjSUS2 (Fig. 4, A–H), suggesting that the LjSUS2a transcript might be less stable.
Our expression analysis showed that the genes encoding the different isoforms of Suc synthase are differentially expressed in L. japonicus (Fig. 4, A–H), as has been reported for pea, M. truncatula, and Arabidopsis (Craig et al., 1999
Phylogenetic analysis of Suc synthases (Fig. 3) showed a clear legume cluster of Suc synthase isoforms as well as a monocot (rice SUS5 and SUS6) and a dicot (LjSUS5 and LjSUS6 and AtSUS5 and AtSUS6) cluster in the "New Group" (Komatsu et al., 2002 To analyze the role of Suc synthase in the nodule, we concentrated on mutations in genes encoding isoforms 1 and 3 because their expression was high in this organ (Fig. 4G). The greatest effects on Suc synthase activity were observed in mutants bearing premature stop codons in the SUS genes (Fig. 6). sus1-1 and sus3-1 were good candidates for being null alleles for the production of the respective SUS enzymes. Line sus3-1 showed a 67% reduction in Suc synthase activity in its nodules, as well as a decrease in the Suc synthase protein content on western blots (Fig. 8). This reduction was sufficient to affect the Suc content of the nodule: Suc levels were elevated in sus3-1 nodules. These results are consistent with more than a 60% contribution of LjSUS3 to total Suc synthase transcript levels in this organ (Fig. 4G). The sus1-1 allele showed a 38% decrease in Suc synthase activity in nodules, consistent with the estimate of LjSUS1 transcript in nodules being about 34% of the total SUS transcript (Fig. 4G). In leaves, the enzyme activity in sus1-1 plants dropped to 50% of wild-type levels, in accordance with the 60% contribution of LjSUS1 to Suc synthase transcript in this organ (Fig. 4, A and B). It was difficult to observe an aberrant phenotype of sus mutants. Signs of nitrogen starvation in sus3-1 mutants were found when plants were grown without nitrogen in CERs, but were not observed for plants grown without nitrogen under glasshouse conditions. sus3-1 mutants grown in the CER started to differ from the wild-type and sus1-1 plants relatively late in growth, after 47 dpi (Table III). After this stage, sus3-1 mutants showed obvious signs of nitrogen starvation: fewer expanded leaves, fewer branches (Table III), yellow basal leaves with reduced chlorophyll content (Fig. 9A; Table IV), reduced fresh weight above ground, and decreased seed fresh and dry weight compared with wild-type plants (Table IV). There was no difference between sus3-1 and wild-type plants with respect to height (Table III), numbers of flowers, or the time of flower and pod formation (data not shown). The glasshouse and the CER differed in temperature, light quality, and intensity, as well as humidity and water saturation of the growth medium. One or several of these differing environmental conditions might explain why nitrogen starvation of sus3-1 mutants is only observed in the CER. We are investigating which factors might be responsible for the induction of nitrogen starvation. sus1-1 mutants did not show signs of nitrogen starvation, which is in agreement with the lower contribution of LjSUS1 to Suc synthase transcript in the nodule (Fig. 4G). Overall, our results showed that LjSUS3 was necessary for normal growth in the CER; LjSUS1 alone could not maintain an adequate level of nitrogen fixation. However, in the glasshouse, LjSUS1 alone could maintain normal growth and LjSUS3 was dispensable.
We also examined the morphology of the wild-type and sus nodules at the peak of LjSUS3 expression (Flemetakis et al., 2006 Results from sus1-1/sus3-1 mutants grown in the CER showed that the aberrant phenotype in these plants was stronger than in sus3-1 mutants (Fig. 9A). These plants lacked 94% of Suc synthase enzyme activity in the nodule and developed an aberrant phenotype at 38 dpi. In contrast to sus3-1 mutants, the sus1-1/sus3-1 double mutants showed significantly reduced height compared with their corresponding wild-type plants at 38 dpi. Plants supplied with nitrogen did not show any signs of nitrogen starvation (Fig. 9B). The more severe phenotype of double mutants confirmed that LjSUS1 contributes to the ability of L. japonicus plants to assimilate nitrogen. Hence, analysis of sus1-1 and sus3-1 lines showed that both the nodule-enhanced isoform LjSUS3 and LjSUS1 can contribute to nitrogen fixation in the L. japonicus nodule. Interestingly, the biomass of the double mutant in the presence of nitrogen was very similar to the wild type, indicating that only the lack of nitrogen was responsible for the difference in growth. Hence, lack of these two isoforms was not significantly impairing the carbon supply to the rest of the plant.
Taken as a whole, our data indicate that invertases cannot metabolize the available Suc to compensate for the loss of Suc synthase activity. A role for nodule-enhanced alkaline/neutral invertase, LjINV1, in nodule function has been proposed by Flemetakis et al. (2006) Prior to our work, the only Suc synthase mutant available in legumes was the rug4 mutant of pea, which lacks the major nodule isoform PsSUS1. Comparison of the rug4 phenotype with that of the L. japonicus mutants allows us to draw the following conclusions, which may well be applicable to legumes generally, namely: both pea and L. japonicus require Suc synthase activity in their root nodules for normal levels of nitrogen fixation; activity of invertases cannot compensate for the role played by Suc synthase in Suc metabolism in either species; and a reduction of total Suc synthase activity in nodules to levels of around 10% or less of those in wild-type plants induces a severe nitrogen starvation phenotype. The relative importance of different isoforms of SUS in contributing to Suc synthase activity in nodules differs between pea and L. japonicus; LjSUS3 is less dominant (68%) than the equivalent isoform (PsSUS1) in pea (89%). Nodules of the sus3-1 mutant do not show premature senescence like their counterparts in PsSUS1 (rug4) mutants. Furthermore, the growth of the double sus1-1/sus3-1 mutant is considerably more impaired than that of either single mutant. Hence, both the nodule-enhanced isoform LjSUS3, as well as LjSUS1, are important for nitrogen fixation in L. japonicus. It will be interesting to discover whether pea is exceptional in the high level of dependence on a single isoform of Suc synthase for carbon utilization by nodules or whether other legumes also display this phenomenon. We have shown that TILLING in L. japonicus is a powerful tool for systematically dissecting the contribution of each single isoform of Suc synthase to nodule metabolism. Whether L. japonicus nodules rely solely on Suc synthase for their supply of ATP, carbon precursors, and reductant or whether the nodule-enhanced alkaline/neutral invertase has a role early in nodule induction, remains to be established. We are currently examining TILLING mutants to determine the role of invertases in the nodule.
Plant Growth Conditions
Plants for RNA extracts were grown in the glasshouse on a Terragreen:sand mixture (1:1; Oil-Dri Ltd.). Plants for enzyme assays, western blots, and GC-MS analysis were grown in the glasshouse on perlite:vermiculite (1:1). Seeds for growth experiments were scarified, sterilized with 10% (v/v) bleach (containing 1% available chlorine), and incubated in sterile water overnight at 4°C. They were germinated on water agar for 5 d before transfer to the respective growth medium: sterilized perlite:vermiculite mixture (1:1) or Terragreen:sand (1:1). Seedlings were immediately inoculated with Mesorhizobium loti strain Tono. Plants on perlite:vermiculite were fed twice a week either with nitrogen-free nutrient solution (Broughton and Dilworth, 1971
LjSUS1
LjSUS2
LjSUS3
LjSUS4
LjSUS5
LjSUS6 Sequences for LjSUS1 to LjSUS6 have been deposited in GenBank with the following accession numbers: AP004481, AP009335, AP009336, AP007582, AP009338, and AP009337, respectively.
Suc synthase gene structures from start to stop codons were predicted using the CODDLE program (http://www.proweb.org./coddle). The ClustalW program (http://www.ebi.ac.uk/clustalw) was used for determining the percent identity between Suc synthase genes.
Protein sequences for 43 Suc synthases were aligned using the ClustalW program. The ends of the alignment (at amino acid 877) were trimmed and seven insertions specific to particular species were removed before performing phylogenetic analysis with Phylip programs (version 3.65). A distance matrix method employing the Dayhoff PAM matrix model was used to compare the sequences. The tree was built using the neighbor-joining clustering method and midpoint rooted as described in the Phylip documentation. Bootstrap values were calculated by analyzing datasets 1,000 times to indicate the confidence of each tree clade.
Plant material was harvested and ground in liquid nitrogen. Total RNA was extracted with CONCERT reagent (Invitrogen) and purified using the RNeasy kit (Qiagen) with on-column DNase I treatment (Qiagen) as described by the manufacturer. Genomic DNA-specific primers were used as controls to confirm the absence of genomic DNA in the RNA extract. RT was performed with 5 µg of RNA using SuperScript reverse transcriptase (Invitrogen) and oligo(dT)15 primers (Promega) according to the manufacturer's instructions.
All samples were measured in technical triplicates on three biological triplicates, consisting of three single plants harvested and extracted individually. Two controls lacking cDNA were included on each 96-well microplate (MJ Research). Master mixes were prepared; each sample contained SYBR Green JumpStart Taq ReadyMix (Sigma), 10 ng cDNA, and gene-specific primers (200 nM). Actin (for flowers) and elongation factor-1
PCR reactions were performed on an Opticon machine (MJ Research). The PCR program started at 94°C for 2 min, and this was followed by 40 cycles of incubation at 94°C (15 s), 60°C (30 s), 72°C (1 min), and 76°C (1 s). Melting curves were recorded from 65°C to 95°C, reading every 0.5°C. For quantification of gene expression, the method described by Livak and Schmittgen (2001
TILLING was carried out to isolate mutants for genes LjSUS1 to LjSUS4 using the general TILLING population and the preselected nodule mutant population described previously (Perry et al., 2003
Genomic DNA isolated from the forward screen plant population and general TILLING population plants were quantified and diluted to 5 ng µL–1. Normalized DNA was pooled 4-fold and 1 µL pooled DNA was used in a 10-µL PCR reaction using gene-specific labeled primers and a touchdown PCR program as described (Colbert et al., 2001
Individuals from a pool containing a positive sample were rescreened to identify the individual plant containing the mutation. The individual was then sequenced and the effect of the mutation predicted using the PARSESNP program as described in Taylor and Greene (2003)
Cleavage Direction
Synthesis Direction
Nodules were harvested and extracted as described for the Suc synthase assay in the cleavage direction. Alkaline and acid invertase activities were assayed as described by Hill et al. (2003)
One root system per sample was placed in a blood tube (Vacutainer; Becton-Dickinson) and sealed with a rubber lid. Acetylene (0.5 mL) was injected and samples were incubated for 2 h at room temperature. The amount of ethylene produced was quantified by comparison with an ethylene standard. A gas chromatograph equipped with a hydrogen flame ionization detector was used. The column was 1.5 m long and 3.25 mm in diameter and packed with Porapak N.
Nodule extracts were prepared as described for the Suc synthase assay (cleavage direction). PAGE and western blotting were performed as described by Barratt et al. (2001)
Gas isotope analysis was carried out using a ThermoFinnigan (GmbH) system with a Conflo III interface. Approximately 10 mg of freeze-dried leaf material was weighed into tin capsules, which were sealed and placed inside a zero-blank autosampler attached to a Costech Elemental Analyzer ECS4010 (Costech International). Samples were analyzed according to Vallet et al. (1991)
Approximately 100 mg of lower leaves were harvested and ground in liquid nitrogen. After addition of 10 mL of 80% (v/v) Tris-buffered acetone (pH 8.0), samples were centrifuged for 5 min at 1,500g. The absorbance at 663.6 nm and 646.6 nm of the supernatant was measured and chlorophyll content calculated following the equations by Porra (2002)
Exactly 100 dry, mature seeds were counted and the fresh weight determined. For seed dry weight, mature seeds were dried at 80°C until seed weight was stable.
Nodules were harvested, immediately frozen in liquid nitrogen, and freeze dried for 24 h. Approximately 14 mg of sample were ground in liquid nitrogen and extracted with 70% (v/v) ethanol according to a protocol by Kadlec (2001)
All samples were measured as a minimum of biological triplicates. Student's t test was used to analyze the statistical significance of the difference between mutant and wild-type data. For qRT-PCR data, the method of Livak and Schmittgen (2001)
The following materials are available in the online version of this article.
We thank Dr. Paul Barratt for the kind gift of antibodies against pea Suc synthase, Alan Jones for GC-MS analysis, and Paul Bailey for assistance with phylogeny. We also thank Jillian Perry for help with the TILLING platform and Andrea Pitzschke for providing EST sequences for some of the LjSUS genes. We are grateful to Professors Cathie Martin and Alison Smith and to Dr. Claire Domoney for helpful comments on the manuscript. Received February 1, 2007; accepted April 19, 2007; published April 27, 2007.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Trevor L. Wang (trevor.wang{at}bbsrc.ac.uk).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.097063 * Corresponding author; e-mail trevor.wang{at}bbsrc.ac.uk; fax 44 1603 450014.
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ABSTRACT
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15N analysis showed that nitrogen fixation was low or absent and nodules senesced early (Craig et al., 1999
or actin in the case of flowers. Values are the means ± SD from three individual plants. Note the asymmetrical distribution of the SD caused by conversion of an exponential process into a linear comparison (Livak and Schmittgen, 2001
method. Methods 25: 402–408
