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Keywords:

  • NLA;
  • ubiquitin ligase with SPX domain;
  • nitrogen limitation;
  • adaptability;
  • AtUBC8;
  • protein–protein interaction

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Abundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In plants, nitrogen has two major roles. First, nitrogen markedly affects plant biomass and crop yield as an essential macronutrient (Lam et al., 1996). Second, as an important signal, nitrogen modulates a number of aspects of plant development including root branching and development, leaf growth, shoot branching and flowering time (Crawford and Forde, 2002), and also regulates the expression of many genes involved in nitrogen and carbon metabolism (Crawford, 1995; Stitt, 1999). Because nitrogen constitutes 1.5–2% of plant dry matter and is a key component of most important macromolecules such as proteins, nucleic acids and chlorophyll, plants must acquire abundant nitrogen nutrient from the soil to achieve optimal growth and development (Marschner, 1995). However, plants are frequently subjected to growth conditions where nitrogen is limiting due to a variety of abiotic and biotic factors such as soil erosion, rainwater leaching and microbial consumption, which markedly reduce the availability nitrogen from the soil (Good et al., 2004). Therefore, adaptability to nitrogen limitation is an essential survival strategy for plants to successfully finish their life cycles and produce offspring, rather than dying early and barren in the presence of limited nitrogen nutrient. In crops, it has been demonstrated that enhancing adaptability to nitrogen limitation could maintain or increase crop yields with reduced application of nitrogen fertilizer. Tollenaar and Wu (1999) reported that increasing the tolerance of maize cultivars to nitrogen limitation made a significant contribution to the genetic improvement of maize yields over the last several decades. Many studies have found that newer released maize hybrids could grow more vigorously and produce higher yields than older ones when grown under conditions of nitrogen limitation, indicating that the newer maize hybrids have a stronger adaptability to nitrogen limitation than do older ones (Castleberry et al., 1984; Ding et al., 2005; Duvick, 1984, 1997; McCullough et al., 1994). Strengthening the adaptability of crop cultivars to nitrogen limitation is important for current agricultural practice, in which large amounts of nitrogen fertilizers are routinely applied to increase crop yields worldwide (Frink et al., 1999). Because 50–70% of the applied nitrogen cannot be absorbed by crops (Peoples et al., 1995), the use of large amounts of nitrogen fertilizer inevitably increases the cost of crop production and also leads to a significant level of nitrogen pollution in the environment (Good et al., 2004). One effective way to overcome these shortcomings is to genetically enhance the adaptability of crops to nitrogen limitation, for which delineation of the molecular mechanism governing this trait is essential (Ding et al., 2005). It is also important to systemically study the physiological and biochemical changes specifically involved in the adaptation of plants to nitrogen limitation.

Plants are able to acclimate to nitrogen limitation by developing various adaptive responses. A number of studies on the effect of nitrogen stress on plant growth and development have been done, and from these some adaptive responses to nitrogen limitation can be inferred. These include the reduction of growth and photosynthesis, remobilization of nitrogen from old, mature organs to actively growing ones, and an accumulation of abundant anthocyanin (Bongue-Bartelsman and Phillips, 1995;Chalker-Scott, 1999; Diaz et al., 2006; Ding et al., 2005; Geiger et al., 1998; Khamis et al., 1990; Mei and Thimann, 1984; Ono et al., 1996). In addition, the expression of some plant genes was found to be specifically regulated by nitrogen limitation. For example, in Arabidopsis the transcript of NRT2.1, a high-affinity nitrate transporter, was increased significantly by nitrate limitation (Filleur et al., 2001), and Todd et al. (2004) found that the expression of a MYB-like gene AtNsr1 was markedly and specifically upregulated by nitrogen deficiency. Recently, Diaz et al. (2006) reported that growing Arabidopsis plants under low-nitrogen conditions resulted in the breakdown of chlorophyll in old rosette leaves and accumulation of anthocyanin in the whole rosette. Fifteen quantitative trait loci (QTL) were identified as being involved in these growth responses caused by nitrogen limitation (Diaz et al., 2006). However, no mutant defective in developing the adaptive responses to nitrogen limitation has been found. Consequently, nothing is known about the molecular mechanism controlling this trait.

In this study, we isolated and characterized an Arabidopsis mutant called the nitrogen limitation adaptation (nla). Supplied with limiting nitrogen nutrient, the nla mutant failed to develop essential adaptive responses to nitrogen limitation but exhibited an early senescence phenotype. The gene responsible, NLA, was identified through a map-based cloning approach. The presence of a RING domain in NLA and its capacity to interact with the Arabidopsis ubiquitin conjugase 8 (AtUBC8) indicate that NLA is a RING-type ubiquitin ligase. In the nla mutant, the RING domain was deleted from the NLA protein, which resulted in the impairment of the interaction between NLA and AtUBC8, the alteration of NLA subcellular localization and disruption of the adaptation of the nla plants to nitrogen limitation. All the results demonstrate that the ubiquitination pathway involving the NLA protein is required to develop the adaptability of Arabidopsis to nitrogen limitation.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Defining nitrogen-limited growth conditions for Arabidopsis plants

The major inorganic nitrogen compound available to crop plants under most soil conditions is nitrate, which is required in great abundance for optimal plant growth and development (Crawford, 1995; Crawford and Forde, 2002). It has been shown that when nitrate is insufficient, plants can develop a set of adaptive responses to the nitrogen-limited growth condition (Chalker-Scott, 1999; Diaz et al., 2006; Ding et al., 2005; Khamis et al., 1990; Mei and Thimann, 1984; Ono et al., 1996;). In Arabidopsis, Bi et al. (2005) found that 10 mm nitrate provided sufficient nitrogen nutrient for Arabidopsis plants, while 3 mm nitrate significantly limited the growth of Arabidopsis plants. To determine what nitrate concentration is suitable for inducing Arabidopsis to develop adaptive responses to nitrogen limitation, we grew Arabidopsis wild-type plants in nitrogen-free soil supplemented with 0.5, 1, 3 and 10 mm nitrate. Consistent with the finding by Bi et al. (2005), 10 mm nitrate was sufficient for the growth and development of Arabidopsis plants throughout their lifetime, while 0.5–3 mm nitrate became limiting at different developmental stages (Figure S1). At the vegetative stage from 18 to 22 days after germination (DAG), the plants grown with 0.5 and 1 mm nitrate had much smaller rosette sizes than those supplied with 10 mm nitrate, and their rosette leaves became dark green, indicating the accumulation of anthocyanin (Figure S1). The plants grown with 3 mm nitrate closely resembled those supplied with 10 mm nitrate in growth and development at the vegetative stage. However, at 26 DAG (about 4–5 days after bolting), these plants reduced their growth markedly and their leaves turned dark green in comparison with the plants grown with 10 mm nitrate (Figure S1). These results indicate that 0.5 and 1 mm nitrate drastically limit the vegetative growth of Arabidopsis plants, and actually subject these plants to severe nitrogen stress. In contrast, 3 mm nitrate provides sufficient nitrogen for vegetative growth of Arabidopsis, but becomes limiting at the generative stage. Consequently, these Arabidopsis plants have well-established rosettes that gradually develop adaptive responses to nitrogen limitation such as reduction in growth and accumulation of anthocyanin (Figure S1). Therefore, application of 3 mm nitrate to nitrogen-free soil provides a suitable growth system for inducing adaptive responses to nitrogen limitation in Arabidopsis plants.

Identification of the nla mutant displaying the early senescence phenotype induced by low inorganic nitrogen

More than 200 T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center (ABRC) seed stocks to isolate mutants with altered growth responses to nitrogen limitation through a reverse genetics approach (Alonso et al., 2003). A collection of 180 homozygous T-DNA insertion lines were identified and grown in nitrogen-free soil with 3 and 10 mm nitrate, respectively, to evaluate their growth performance. One T-DNA insertion line failed to adapt to the conditions of nitrogen limitation and started senescence much earlier and more rapidly than did wild-type plants supplied with 3 mm nitrate (Figure 1). Accordingly, this T-DNA insertion line was called the nitrogen limitation adaptation (nla) mutant. Figure 1(a–c) shows that the nla mutant plants supplied with 10 mm nitrate had a similar pattern of growth and development to wild type. When the nitrate concentration was reduced to 3 mm, the nla plants started senescence in the fifth rosette leaf at 24 DAG, and after this point senescence progressed rapidly with all rosette leaves showing senescence symptoms at 26 DAG, and the whole rosette dying at 32 DAG. In contrast, wild-type plants displayed no senescence symptoms in the fifth rosette leaf until 32 DAG. In wild-type plants, the process of senescence proceeded slowly and gradually from the fifth to the youngest rosette leaves, and it took at least 2 weeks for all rosette leaves to show the senescence symptoms. The cauline leaves in the nla plants started senescence at 28 DAG, at least 10 days earlier than those in the wild-type plants (Figure 1d). Further, the developing nla siliques initiated senescence in their tips at 32 DAG, while the wild-type siliques showed no senescence symptoms throughout their development but accumulated abundant anthocyanin which was not observed in the nla siliques (Figure 1e). With the reduction of the nitrate concentration to 1 mm, the occurrence of the senescence phenotype in the rosette leaves of nla plants was accelerated to 20 DAG, and severe senescence in the developing nla siliques resulted in their death around 30 DAG without producing viable seeds. Under the same growth condition (1 mm nitrate), wild-type plants did not start senescence in their rosette leaves until 26 DAG, and produced fecund siliques (Figure 1c).

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Figure 1.  The low-nitrogen-induced early senescence phenotype in the nla mutant. Wild-type (Columbia, Col) and nla plants were grown with 1, 3 or 10 mm nitrate for 18 days (a), 26 days (b) and 32 days (c), showing the early senescence phenotype in the nla plants supplied with 1 or 3 mm nitrate. DAG, days after seed germination. Arrows indicate the dead siliques. Parts (d) and (e) show cauline leaves and developing siliques, respectively, from the nla (upper panel) and Col (lower panel) plants supplied with 3 mm nitrate. The arrow in (e) indicates the senescing silique tip. Supplying senescing nla plants with 15 mm nitrate stopped the progress of senescence in the nla plants initially grown with l mm (f) or 3 mm (g) nitrate.

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To further confirm that the early senescence phenotype in the nla mutant is dependent on insufficient nitrate supply, we grew nla plants with 1 or 3 mm nitrate. When the symptoms of senescence were just initiated in the fifth rosette leaves, these plants were supplied with 15 mm nitrate. Subsequently, the process of senescence in the senescing nla plants was stopped, with the senesced rosette leaves renewing their growth and new rosette leaves being free of symptoms of senescence. Further, new lateral shoot branches were produced, and no symptoms of senescence were seen in the cauline leaves or the siliques (Figure 1f,g). Finally, siliques in these nla plants became fecund (Figure 1f) rather than dying (Figure 1c) after they were supplied with 15 mm nitrate.

Besides nitrate, other inorganic nitrogen fertilizers include ammonium and ammonium nitrate. When grown with 5.0 mm ammonium or 2.5 mm ammonium nitrate, nla plants started a rapid process of senescence in the rosette leaves around 24 DAG, while wild-type plants showed no symptoms of senescence until 32 DAG (data not shown), indicating that limiting ammonium and ammonium nitrate supply also induces the early senescence phenotype in the nla mutant. Since the three inorganic forms of nitrogen have the same effect on the nla mutant, we used nitrate as the nitrogen source in the following experiments.

To determine whether the nla mutant would produce the early senescence phenotype with low phosphorus application, both wild-type and nla plants were grown in phosphorus-free soil supplemented with 0.5, 1 and 10 mm phosphate. After 26 DAG, both wild-type and nla plants supplied with 0.5 and 1 mm phosphate showed obvious small rosette sizes and dark green leave as compared with those grown with 10 mm phosphate (Figure 2a–c). This observation indicates that 0.5 and 1 mm phosphate are insufficient for wild-type and nla plants, but cannot induce the early senescence phenotype in the nla plants. Further, the nla and wild-type plants grown with full nutrients for 18 days were treated with drought and high temperature stress for 8 and 10 days, respectively, and both genotypes showed a similar pattern of growth and development (Figure 2d,e). These results suggest that the nla mutant is specifically sensitive to the application of nitrogen, and exhibits the early senescence phenotype only under nitrogen-limited growth conditions.

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Figure 2.  The effect of three different stresses on the growth and development of the nla and wild-type plants (Columbia, Col). (a–c) The nla and wild-type plants grown with 10 mm (a), 1 mm (b) and 0.5 mm (c) phosphorus for 26 days. (d, e) The nla and wild-type plants were grown with full nutrients for 18 days and then treated with high-temperature stress (d) or drought (e).

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The nla mutant did not alter its capacity for acquiring nitrogen but instead had an altered senescence process mediated by nitrogen limitation

There are two possible explanations for why the nla mutant has an early senescence phenotype induced by low inorganic nitrogen. First, nla plants may acquire less nitrogen nutrient than wild-type plants. To address this issue, we examined the total nitrogen content in wild-type and nla plants. Supplied with high (10 mm) or low (3 mm) nitrate, the wild type and the nla plants at 18 DAG resembled each other in fresh weight (data not shown) and total percentage of nitrogen (Figure 3a). Thus, the nla mutant closely resembles wild type with respect to total nitrogen content. Further, the major nitrate transporter genes, NRT1.1 (low nitrate affinity transporter) and NRT2.1 (high nitrate affinity transporter), had very similar levels of expression in the roots of wild-type and nla plants supplied with 3 or 10 mm nitrate (Figure 3b). These results suggest that nla and wild-type plants have a similar capacity to acquire nitrogen nutrient no matter whether the nitrogen supply is high or low.

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Figure 3.  Comparison of the nitrogen acquisition and senescence process in nla and Col plants grown with limiting nitrogen nutrient. (a) Total percentage nitrogen content (w/w) in shoots at 18 days after seed germination (DAG). The values are means ± standard error (= 3). (b) Expression of two major nitrate transporter genes NRT1.1 and NRT2.1 in nla and Col roots at 20 DAG. (c) Progress of senescence in rosette leaves from the NLA and Col plants at 26 DAG (upper panel) and 32 DAG (lower panel). Photographs show representative leaves at each position in a rosette. (d) The expression pattern of SAG12, CAB and RBCS in nla and Col plants. Ubiquitin was used as a cDNA synthesis and amplification control, and amplified from undiluted (Ubiquitin) and fivefold (5 ×) diluted cDNA

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The second possible reason for nla plants to produce the early senescence phenotype only when nitrogen availability is limited may be that nla plants are impaired in developing the adaptive responses that enable Arabidopsis plants to acclimatize to conditions of nitrogen-limited growth. One such adaptive response is nitrogen limitation-mediated senescence, which is essential for remobilizing the nitrogen nutrient from old, mature rosette leaves to young actively growing organs, such as young leaves and immature seeds (Mei and Thimann, 1984; Thimann, 1980). Therefore, the process of senescence mediated by nitrogen limitation in wild-type and nla rosette leaves was examined in detail (Figure 3c). When grown with 3 mm nitrate, wild-type plants showed no symptoms of senescence in the fifth and younger rosette leaves until 32 DAG. Senescence progressed slowly and was well organized, as indicated by the observation that the rosette leaves gradually changed their colour from green to dark green and red, and kept their turgidity throughout the senescence process. In the nla plants supplied with 3 mm nitrate, senescence not only started much earlier than that in wild type, but also progressed very rapidly because all the rosette leaves showed symptoms of senescence at 26 DAG, and displayed abrupt change in leaf colour and swift disappearance of leaf turgidity. The lower leaf blades of the senescing leaves were still green and turgid, while the upper parts had already died (Figure 3c). Finally, the senescing leaves did not turn to red from green, and the dead leaves were brown with some redness, indicating little increase in anthocyanin content during the senescence process in the nla plants.

The Arabidopsis Senescence Associated Gene 12 (SAG12) is a gene associated with senescence and has been defined as an authentic molecular marker of senescence (Noh and Amasino, 1999). We determined the expression of SAG12 in the fifth to eighth rosette leaves of wild-type and nla plants growing with 3 mm nitrate to make it more convenient and accurate to track the occurrence of senescence and sample rosette leaves for biochemical assays. As shown in Figure 3(d), expression of SAG12 was detected in the nla plants at 24 DAG and increased with the start of senescence at 28 DAG. On the other hand, SAG12 in wild-type plants was not expressed until 32 DAG, and increased markedly at 36 DAG when severe senescence occurred in the rosettes. These results indicate that the level of expression of SAG12 indeed reflects the senescence process in the rosette leaves, and thus justified the use of SAG12 expression to track the occurrence of senescence and to sample rosette leaves for biochemical assays.

The nla mutant contained high levels of nitrogen metabolites while failing to accumulate soluble sugars and starch in the senescing rosette leaves

To make full use of the available nitrogen nutrient under conditions where it limits growth, plants can export nitrogen from old leaves and organs to young and developing ones. Therefore, the second adaptive response to nitrogen limitation that we tested is the remobilization of nitrogen from senescing leaves. The rapid senescence and death of rosette leaves in nla plants may impair the remobilization of nitrogen from these senescing leaves to young leaves, flowers and developing siliques. If this is the case, it would result in a high content of nitrogen metabolites in the nla rosette leaves. To test this hypothesis, the levels of nitrate, amino acids, soluble proteins and total nitrogen were determined in the fifth to eighth rosette leaves of wild-type and nla plants grown under nitrogen-limited conditions throughout the senescence process. At 18 DAG, prior to the initiation of senescence, wild-type and nla plants contained very similar amounts of the three nitrogen-containing compounds (Figure 4a–c) and did not differ significantly in total nitrogen content (Figure 4d). At 32 DAG, when senescence occurred in wild-type rosette leaves, the content of nitrate, total amino acids, soluble proteins and total nitrogen was reduced by 75%, 80%, 75% and 70%, respectively, as compared with those at 18 DAG when no symptoms of senescence were observed. With the progression of senescence in the wild-type rosette leaves at 36 DAG, the nitrate, total amino acids, soluble proteins and total nitrogen content was decreased by 90%, 90%, 90% and 80%, respectively (Figure 4a–d). In contrast, the occurrence (at 24 DAG) and progression (at 28 DAG) of senescence in nla rosette leaves were not accompanied by a significant reduction in the amounts of nitrate, total amino acids, soluble proteins and total nitrogen (Figure 4a–d). For example, when severe senescence occurred in nla rosette leaves at 28 DAG, the content of nitrate, total amino acids, soluble proteins and total nitrogen was only decreased by 33%, 5%, 20% and 6%, respectively, as compared with that at 18 DAG (Figure 4a–d). These results suggest that nitrogen is remobilized from senescing wild-type rosette leaves, while this did not occur to nearly the same extent in the nla senescing leaves.

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Figure 4.  Biochemical changes throughout the senescence process in wild-type (Col) and nla plants grown with 3 mm nitrate. The metabolites assayed are: (a) nitrate; (b) total amino acids; (c) proteins; (d) total nitrogen percentage (w/w); (e) glucose; (f) fructose; (g) sucrose; (h) starch; (i) anthocyanin; (j) chlorophyll. Data are the mean values ± standard deviation (= 3–6).

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Nitrate assimilation involves the use of large amounts of organic acids from photosynthesis to synthesize amino acids. Low nitrogen availability decreases the flow of organic acids into amino acids, and more organic acids are diverted to the production of sugars and starch. Therefore, the accumulation of starch and soluble sugars, including glucose, fructose and sucrose, in leaves has been found to be strongly linked to the occurrence of nitrogen deficiency and leaf senescence (Diaz et al., 2005; Paul and Driscoll, 1997; Wingler et al., 2006). As shown in Figure 4(e–h), glucose, fructose, sucrose and starch accumulated markedly with the start and progression of leaf senescence in wild-type plants, but the content of the three soluble sugars and starch in the nla plants was only slightly increased when senescence occurred in their rosette leaves. For example, at 18 DAG when leaf senescence had not been initiated, wild-type and nla plants had similar levels of glucose, fructose, sucrose and starch. When leaf senescence occurred, the level of glucose, fructose, sucrose and starch increased 90%, 84%, 46%, and 18-fold, respectively, in wild-type plants (at 36 DAG), while only increasing 5%, 8%, 20% and 2.5-fold, respectively, in the nla plants (at 28 DAG).

The nla mutant was impaired in the accumulation of anthocyanin and the reduction of photosynthetic capacity during senescence

Accumulating anthocyanin and reducing photosynthesis are two important adaptive responses to nitrogen limitation, and are controlled by multiple QTLs in Arabidopsis (Diaz et al., 2006). To determine whether the nla plants could develop such adaptive responses, the amount of anthocyanin and the photosynthetic capacity were measured in wild-type and nla plants. Wild-type plants grown under limiting nitrogen increased their anthocyanin content markedly during growth and senescence (Figure 4i). At 18 DAG, the fifth to eighth rosette leaves of wild-type plants contained 4.8 U g-1 fresh weight (gFW−1) of anthocyanin, and this increased to 31 U gFW−1 at 28 DAG when rosette leaves still showed no symptoms of senescence. With the occurrence of senescence in the fifth to eighth rosette leaves of wild-type plants at 32 DAG, their anthocyanin content increased to >40 U gFW−1. In contrast, anthocyanin accumulation did not occur in the senescing nla rosette leaves (Figure 4i). At 18 DAG, when the nla plants showed no molecular or morphological symptoms of senescence, they contained 4.0  U gFW−1 anthocyanin. At 24 DAG, when senescence occurred in the fifth to eighth rosette leave, they still contained 3.5  U gFW−1 anthocyanin. No increase in the anthocyanin content was observed when all the rosette leaves showed severe senescence symptoms in the nla plants at 28 DAG (Figure 4i).

It has been shown that the photosynthetic capacity of plants is correlated with chlorophyll content and the expression of two photosynthesis marker genes RBCS and CAB in leaves, which encode the small subunit of Rubisco and the chlorophyll a/b binding protein, respectively (Martin et al., 2002). The chlorophyll content was assayed in wild-type and nla plants supplied with limited nitrogen. As shown in Figure 4(j), wild-type and nla plants contained 0.99 and 0.95 mg chlorophyll gFW−1, respectively, at 18 DAG. With the initiation (32 DAG) and progress (36 DAG) of senescence in wild-type plants, the chlorophyll content was decreased by 50% and 70%, respectively. However, the chlorophyll content was only reduced by 15% at 28 DAG when severe senescence occurred in the nla plants. Reverse transcriptase-PCR analysis revealed that the expression of RBCS and CAB was reduced drastically when the low-nitrogen-mediated senescence occurred in wild-type rosette leaves at 32 DAG (Figure 3d). In contrast, no significant reduction in RBCS and CAB expression in nla plants accompanied senescence occurring in their rosette leaves (Figure 3d). These data indicate that, unlike wild-type plants, nla plants grown with limited nitrogen nutrient are impaired in the accumulation of anthocyanin and in the reduction of photosynthetic capacity, which are essential adaptive responses to nitrogen limitation.

Genetic characterization of the nla mutant and map-based cloning of the NLA gene

To determine the inheritance of the low inorganic nitrogen-induced early senescence phenotype in the nla mutant, we backcrossed this mutant to wild type. The F1 plants showed the same phenotype as wild-type plants when grown on 3 mm nitrate. In the F2 generation, wild-type and nla mutant phenotypes segregated at a ratio of 3:1 (data not shown), indicating that the nla mutant phenotype is recessive and inherited as a single Mendelian trait. Southern blot analysis revealed that the genome of the nla mutant contained five T-DNA insertions, while none was genetically linked to the early senescence phenotype induced by limited nitrogen (data not shown), indicating that the responsible gene in the nla mutant was not tagged by a T-DNA insertion. Subsequently, the nla mutant was successively backcrossed to wild type four times, with no T-DNA insertion being left in the nla mutant.

Given that the NLA gene is not tagged by a T-DNA insertion in the nla mutant, a map-based cloning approach was used to isolate NLA. An F2 mapping population was generated by crossing the nla mutant with Landsberg erecta wild type. Genomic DNA from 50 F2nla mutant plants was combined and used for the initial mapping with the 22 simple sequence length polymorphism (SSLP) markers from Lukowitz et al. (2000). A close linkage was detected between NLA and the SSLP marker F21M12 on the top arm of chromosome 1 (data not shown). Fine mapping with more SSLP markers and cleaved amplified polymorphic sequence (CAPS) markers located the NLA locus to a genomic region between the SSLP marker 473993 and the CAPS marker SNP247 on the bacterial artificial chromosome (BAC) clone F22D16. This region is approximately 62.3 kb with 21 annotated genes (Figure 5a). Thirteen genes could be the candidate for NLA, and their coding regions as well as the corresponding genomic sequences were amplified from the nla and wild-type plants by PCR. Comparing these PCR products revealed that only the gene At1g02860 carried a deletion in its genomic and coding sequences in the nla mutant. The mutated At1g02860 gene lost its third intron and fourth exon, while the remaining exons 3 and 5 were fused in frame (Figure 5a).

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Figure 5.  Map-based cloning of the NLA gene and molecular analysis of NLA protein from the wild type and the nla mutant. (a) The position of the NLA locus was defined by two flanking SSLP markers NF21B7 (12 recombinants) and NT7I23 (six recombinants) on the top arm of Chromosome 1. Fine mapping located NLA locus on the BAC clone F22D16 flanked by the SSLP marker 473993 (one recombinant) and the CAPS marker SNP247 (one recombinant). This region contained 21 annotated genes, among which DNA fragment deletion was only detected in At1g02860. (b) Complementation testing confirmed At1g02860 as the NLA gene. Three nla plants independently transformed with At1g02860 cDNA did not show the early senescence phenotype when supplied with 3 mm nitrate (upper panel) and contained wild-type At1g02860 mRNA (lower panel). The nla plant transformed with the empty vector pEGAD displayed the early senescence phenotype and did not have wild-type At1g02860 mRNA. (c) The predicted amino acid sequence of NLA protein. The SPX and RING domains are indicated by blue and red ink, respectively. The deleted residues in the truncated NLA protein (nla) of the nla mutant are underlined. (d) Scheme of the NLA protein structure with the SPX (blue) and RING (red) domains.

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No T-DNA insertion line was found in At1g02860 in the ABRC seed stocks. To confirm that At1g02860 is indeed the NLA gene, the wild-type At1g02860 cDNA driven by the 35S promoter was transformed into the nla mutant. Three independent transformants expressing the transformed wild-type At1g02860 cDNA (Figure 5b) did not show the early senescence phenotype when supplied with 3 mm nitrate (Figure 5b). In contrast, the nla plant transformed with the empty binary vector pEGAD (Cutler et al., 2000) still exhibited the early senescence phenotype induced by low nitrate (Figure 5b). Further, we genotyped 233 plants from the F2 population of nla × wild-type cross with two primers which can specifically determine the deletion of At1g02860 in the nla mutant (data not shown). Fifty-five plants showing the low nitrogen-induced early senescence phenotype were homozygous in the deletion, and the other plants with wild-type phenotype were heterozygous in the deletion (116 plants) or had no such deletion (62 plants). Therefore, all the data unambiguously assign At1g02860 as the nla gene, and the mutated At1g02860 gene is responsible for the low-nitrogen-induced early senescence phenotype in the nla mutant.

The NLA gene encodes a RING-type ubiquitin E3 ligase and is expressed throughout the plant

The NLA gene consists of six exons and five introns and encodes a protein of 335 amino acids (Figure 5a,c) with a molecular weight of 38 110 Da and a pI of 4.59. The NLA protein harbours two known domains, RING and SPX (Figure 5c,d). In Arabidopsis, functional characterization of some RING-containing proteins such as COP1 and SINATA5 suggests that the biological function of the RING domain is to participate in ubiquitin-dependent protein degradation (Moon et al., 2004), and thus plays a central and essential role in eukaryotic cellular regulation (Glickman and Ciechanover, 2002). Stone et al. (2005) reported that the Arabidopsis genome encodes 469 putative RING-containing proteins, which can be grouped into eight types, and NLA belongs to the RING-HCa type (Stone et al., 2005). The SPX domain was named after the yeast proteins SYG1 and PHO81 as well as the mammalian protein XRP1, all of which contain this 180-amino-acid domain at their N-terminus (Battini et al., 1999; Spain et al., 1995; Wykoff and O’Shea, 2001). However, the exact biological function of the SPX domain is still not clear. The mutated NLA gene in the nla mutant encodes a truncated NLA protein called nla (Figure 5d). Comparing the amino acid sequence of the NLA protein with that of nla reveals that both proteins have almost identical amino acid sequences except that nla does not contain the RING domain (Figure 5c,d).

Reverse transcriptase-PCR analysis revealed that the expression level of the NLA gene was high in root and stem, medium in seedling, flower, rosette and cauline leaves, and very low in siliques (Figure S2a). To analyse the transcription pattern of the NLA gene in more detail, its promoter was fused with the coding region of the β-glucuronidase (GUS) reporter gene and expressed in Arabidopsis plants. The transcription of the NLA promoter–GUS construct was evident in cotyledons and hypocotyls, but weak in the roots of 5-day-old seedlings (Figure S2b). In the mature plant, GUS staining was very strong in root and stem, followed by rosette and cauline leaves (Figure S2c–e). In flowers, strong GUS expression was detected in pedicel, receptacle, pistil, sepal and the filament of the stamen (Figure S2f). Developing siliques showed GUS staining only at the two ends (Figure S2 g). Although the Arabidopsis root has strong NLA expression, the root growth and morphology of the nla mutant was similar to that of wild type in the medium with low nitrate application for 3 weeks (data not shown).

The NLA protein interacts with Arabidopsis ubiquitin conjugase 8 in nuclear speckles

In a functional analysis of the RING-type ubiquitin ligase family in Arabidopsis, Stone et al. (2005) reported that the NLA protein, when expressed in Escherichia coli and used for in vitro activity assay, failed to show ubiquitin ligase activity. However, we found that the NLA protein was very unstable when expressed in E. coli (data not shown). Therefore, the functionality of the NLA protein as an ubiquitin ligase was analysed by examining its ability to interact with any Arabidopsis ubiquitin conjugase (AtUBC), which is the essential trait for a ubiquitin ligase. In a yeast two-hybrid screen of a cDNA library made from Arabidopsis leaves, the NLA protein was found to interact with AtUBC8 (Figure 6a–d). In contrast, the construct expressing the nla protein which lacks the RING domain failed to interact with AtUBC8, indicating that the RING domain is indispensable for the interaction of NLA with AtUBC8 (Figure 6d).

image

Figure 6.  Interaction between NLA and AtUBC8 shown by yeast two-hybrid analysis (a–d) and bimolecular fluorescence complementation in onion epidermal cells (e). (a) Schematic diagram of the NLA, nla and AtUBC8 constructs used in the yeast two-hybrid analysis. BD is the GAL4-binding domain and AD the GAL4-activation domain. (b) Diagram of the plasmid pairs used in the yeast two-hybrid experiment. The plasmid pair of pGBKT7-53 and pGADT7-SV40 was used as a positive control. (c), (d) Yeast cells transformed with the plasmid pairs were cultured on the control medium (selection medium + histidine) and the selection medium, respectively. (e) Schematic diagram of the protein constructs used to transform onion epidermal cells (upper panel) and the bimolecular reconstitution of the fluorescent YFP (middle panel). The corresponding nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; lower panel).

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A bimolecular fluorescence complementation experiment was performed to confirm the direct interaction between NLA and AtUBC8 (Hu et al., 2002). NLA and AtUBC8 were fused respectively with the N-terminal and the C-terminal regions of the yellow fluorescence protein (YFP), and simultaneously expressed in onion epidermal cells. The interaction between NLA and AtUBC8 reconstituted the fluorescent YFP in the nuclear speckles (Figure 6e). The RINGp protein, which contains the RING domain while lacking the SPX domain, also interacted with AtUBC8 to produce the fluorescent YFP in the nucleus and cytoplasm (Figure 6e). In contrast, no YFP fluorescence was observed in the onion epidermal cells transformed with the constructs encoding AtUBC8 and the nla protein, respectively, indicating that no interaction occurred between AtUBC8 and the NLA protein which lacks the RING domain (Figure 6e).

NLA–GFP (green fluorescence protein) fusion protein was found to be localized to distinct nuclear speckles, and AtUBC8–GFP resided at the nucleus and cytoplasm, which is consistent with the observation that the interaction between NLA and AtUBC8 occurred in the nuclear speckles (Figure 7). Interestingly, both the RINGp (NLA without the SPX domain)–GFP and nla (NLA without the RING domain)–GFP fusion proteins were distributed in the nucleus and cytoplasm (Figure 7), indicating that both the RING and SPX domains are essential for targeting NLA to the right subcellular localization.

image

Figure 7.  The subcellular localization of the different GFP fusion proteins. Upper panel: the structures of NLA:GFP, nla:GFP, RINGp:GFP and AtUBC8:GFP fusion proteins. Middle panel: the subcellular localization of the four GFP fusion proteins with the intact GFP as the control. Lower panel: nuclei stained by DAPI.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth and development involves the consumption of abundant nitrogen nutrient from the soil (Crawford, 1995; Crawford and Forde, 2002). When this high level of nitrogen is unavailable, plants are able to adjust their growth, development and metabolism to adapt their growth to the nitrogen-limited conditions (Chalker-Scott, 1999; Diaz et al., 2006; Ding et al., 2005; Khamis et al., 1990; Mei and Thimann, 1984; Ono et al., 1996). However, the signalling mechanism via which this nitrogen limitation is recognized is entirely unknown. The evidence for the existence of a nitrogen limitation sensing and signalling pathway in living organisms comes from yeast and bacteria. In yeast, the ammonium permease Mep2p can sense the availability of nitrogen in the environment, and subsequently generate the nitrogen limitation signal for yeast to regulate its growth and development to acclimatize to the unfavourable growth conditions (Biswas and Morschhäuser, 2005; Gagiano et al., 2002; Lorenz and Heitman, 1998). In bacteria, the PII-type signal transduction proteins have been found to play a central role in the nitrogen limitation signalling pathway, and their function and interaction with other proteins are modified by phosphorylation, uridylylation or adenylylation in response to the intracellular nitrogen status (deficient or sufficient; Arcondeguy et al., 2001; Schwarz and Forchhammer, 2005). Although the plant orthologue of bacterial PII protein has been identified, it does not have a similar function in regulating adaptability to nitrogen limitation in plants (Moorhead and Smith, 2003). In this study, we presented physiological, biochemical and molecular genetic data to clearly demonstrate that the NLA protein, a ubiquitin ligase, is required for the development of the adaptive responses to nitrogen limitation, and is an essential component in a ubiquitination dependent pathway controlling the adaptability of Arabidopsis to nitrogen limitation.

Nitrogen limitation stress has been known to markedly affect plant growth and development, while knowledge about the adaptability of plants to nitrogen limitation is lacking. In this study, we defined that 3 mm nitrate applied in a nitrogen-free soil provided a nitrogen-limited growth condition for Arabidopsis plants (Figure S1), and subsequently demonstrated that Arabidopsis plants are able to acclimatize to the nitrogen-limited growth condition by developing a set of adaptive responses to nitrogen limitation (Figure S1 and Figure 4). First, the photosynthetic capacity was reduced. This response markedly decreases the plant’s requirement for the nitrogen nutrient, and also restricts the utilization of photosynthates in synthesizing nitrogen-containing molecules, such as amino acids, proteins and nucleic acids. Second, the synthesis of anthocyanin was significantly increased. The increase in this light-protecting pigment allows the nitrogen-deficient plants to avoid photoinhibition damage caused by nitrogen limitation (Bongue-Bartelsman and Phillips, 1995; Chalker-Scott, 1999). Third, soluble sugars and starch, which are strongly linked to nitrogen deficiency and leaf senescence, were accumulated (Diaz et al., 2005; Paul and Driscoll, 1997; Wingler et al., 2006). Leaf senescence has been known to be important for nitrogen recycling in Arabidopsis because >80% of the total nitrogen in mature rosette leaves is exported through the senescence process (Himelblau and Amasino, 2001). Under nitrogen-limited growth conditions, this senescence-mediated nitrogen recycling becomes more important for Arabidopsis plants to efficiently use the limited nitrogen nutrient, and is thus an essential adaptive response to nitrogen limitation. In this study, Arabidopsis plants grown with a limited nitrogen supply began senescence in old mature rosette leaves at the late bolting stage, and senescence progressed gradually from the older mature rosette leaves to younger ones (Figure 3c). Concomitantly, the total nitrogen content and the amount of nitrogen-containing compounds such as proteins, amino acids and chlorophyll decreased markedly in the senescing rosette leaves, indicating that nitrogen in these leaves was exported to young, developing organs such as floral buds and siliques (Figure 4). These physiological and biochemical changes involved in the adaptive responses to nitrogen limitation in Arabidopsis plants suggest that a sensing or signalling pathway for nitrogen limitation is activated to initiate such a complicated metabolic response to low nitrogen availability.

In contrast, the nla mutant failed to acclimate to nitrogen-limited growth conditions, as indicated by its early senescence phenotype induced by nitrogen limitation. When grown with 3 mm nitrate, the nla plants started senescence in rosette leaves at least 1 week earlier than did wild-type plants, while those supplied with sufficient nitrogen nutrient (10 mm nitrate) were similar to wild type in every aspect of growth and development throughout their life cycle (Figure 1a–c). The early senescence phenotype of the nla mutant induced by low nitrate could be explained either by a defect in nitrogen acquisition or by the loss of adaptability to the nitrogen-limited growth conditions. The first possibility was excluded because the nla and wild-type plants had a very similar total nitrogen content when either 3 mm or 10 mm nitrate was supplied (Figure 3a). The second possibility was confirmed by our finding that the physiological and biochemical changes essential for adaptive responses to nitrogen limitation failed to occur in the nla plants supplied with limited nitrate (Figure 4). First, from the late vegetative to the reproductive stage, the nla plants grown with a limited nitrogen supply did not accumulate anthocyanin, and only had a slight reduction in photosynthesis and little increase in soluble sugar and starch content (Figure 4). Second, the total nitrogen content and the nitrogen-containing compounds such as proteins and total amino acids in the senescing nla leaves were only slightly reduced with the initiation and progress of senescence in the nla rosette leaves (Figure 4), indicating that senescence-mediated nitrogen remobilization did not occur in the senescing nla rosette leaves. These results strongly suggest that the mutation in the nla gene may disrupt the sensing of nitrogen limitation or the signalling pathway, so that the nla mutant does not signal the nitrogen-limited growth conditions and fails to alter its physiological and biochemical status accordingly.

The NLA gene whose mutation causes the nla mutant phenotype was identified through a map-based cloning approach and confirmed by the genetic complementation of the nla mutant phenotype (Figure 5). Two known domains, SPX and RING, were identified in the NLA protein. Many eukaryotic proteins such as yeast VTC1–4 proteins, SGY1 and mammalian XPR1 also contain the SPX domain, but its biological function has still not been determined (Battini et al., 1999; Müller et al., 2002; Spain et al., 1995). Functioning in the yeast signalling phosphate starvation pathway, PHO81, PHO87, PHO90 and PHO91 are known to be involved in phosphate transport or sensing, and all have an SPX domain (Wykoff and O’Shea, 2001). In Arabidopsis, PHO1 protein contains an SPX domain and has been found to function in loading root phosphorus into the xylem (Hamburger et al., 2002). These findings suggest that SPX domain-containing NLA protein may also play a role in the phosphorus starvation responses in Arabidopsis. Accordingly, the nla mutant may differ from wild type in responding to low phosphorus application. However, this possibility was excluded by the fact that, supplied with limited phosphorus nutrient, the nla mutant has the same growth and development pattern as the wild type, and does not show the early senescence phenotype (Figure 2a–c).

The presence of a RING domain in NLA suggests that this protein may function as a RING-type ubiquitin ligase (Figure 5). Nevertheless, the instability of the NLA protein expressed in E. coli (data not shown) makes it very difficult to exhibit the ubiquitin ligase activity of NLA in vitro. This is consistent with the report from Stone et al. (2005) that the E. coli-expressed NLA did not have ubiquitin ligase activity. In this study, we demonstrated the ubiquitin ligase functionality of NLA by showing that NLA is able to interact with an Arabidopsis ubiquitin conjugase (AtUBC), which is the essential property of a ubiquitin ligase (Moon et al., 2004). First, NLA interacted with AtUBC8 in a yeast two-hybrid assay, and this interaction was prevented by the deletion of the RING domain from NLA (Figure 6a–d). Second, in a bimolecular fluorescence complementation experiment (Hu et al., 2002), the interaction between NLA and AtUBC8 reconstituted the fluorescent YFP, and the presence of the RING domain in NLA is essential for this interaction to occur (Figure 6e). Third, the interaction of NLA and AtUBC8 occurs in the nuclear speckles, consistent with the subcellular localization of NLA (Figure 6e and 7). Besides conferring the ubiquitin ligase functionality on NLA, the RING domain is also critical for the physiological function of NLA in Arabidopsis. Deletion of the RING domain from NLA in the nla mutant changed the subcellular localization of NLA from the nuclear speckles to the cytoplasm and nucleus (Figure 7), and prevented its interaction with AtUBC8 (Figure 6e). Consequently, the adaptability to nitrogen limitation was disrupted in the nla mutant.

The functionality of NLA as a ubiquitin ligase suggests a functional model for the role of NLA in developing adaptability to nitrogen limitation, given that ubiquitination-dependent degradation or modification of important regulatory proteins in eukaryotes such as nuclear transcriptional factors has been known to play central roles in regulating numerous cellular processes (Glickman and Ciechanover, 2002; Schnell and Hicke, 2003; Sun and Chen, 2004). We propose that NLA may be involved in the ubiquitination-mediated degradation or modification of substrate protein(s), which may function as the key negative regulator(s) in the nitrogen limitation sensing or signalling pathway. In the nla mutant, the negative regulator(s) would not be degraded or modified properly by the nla protein which lacks the RING domain. Consequently, the nla plants grown with limited nitrogen nutrient cannot sense or signal the nitrogen-limiting conditions, and fail to develop the essential adaptive responses to nitrogen limitation.

Crop cultivars with enhanced adaptability to nitrogen limitation are important in all growing regions, but this is particularly desirable in developing countries where farmers cannot afford the cost of buying large amounts of nitrogen fertilizer and where the need to enhance crop productivity is very high (Duvick, 1997; Loomis, 1997). Understanding the molecular mechanism controlling this process could greatly accelerate the development of such crop cultivars. In this study, the cloning and functional characterization of NLA indicates that plants are equipped with a molecular mechanism to adapt to nitrogen limitation, and can be used to identify the molecular components involved in governing the sensing, signalling and associated gene regulation concerned with nitrogen limitation in plants.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth conditions and isolation of the nla mutant

A collection of 180 Arabidopsis homozygous T-DNA insertion mutant lines (in Columbia background) were identified from ABRC seed stocks. In a growth room with controlled environmental conditions (23°C day/18°C night, white fluorescent illumination of 150 µmol m−2 sec−1, 16 h light/8 h dark and 75% relative humidity), these T-DNA lines were grown in the nutrient-free soil LB2 (Sun Gro Horticulture Canada Ltd, http://www.sungro.com/) supplied with 3 or 10 mm potassium nitrate in the nutrient solution (10 mm KH2PO4, 2 mm MgSO4, 1 mm CaCl2, 0.1 mm Fe-EDTA, 50 µm H3BO4, 12 µm MnSO4, 1 µm ZnCl2, 1 µm CuSO4, 0.2 µm Na2MoO4) once a week for 4 weeks. Based on the low-nitrate-induced early senescence phenotype, the nla mutant was isolated from these T-DNA insertion lines. To determine the responses of the nla and wild plants to low phosphorus application, both plants were grown in the LB2 soil supplied with 0.5, 1 and 10 mm KH2PO4 in the nutrient solution (10 mm KNO3, 2 mm MgSO4, 1 mm CaCl2, 0.1 mm Fe-EDTA, 50 µm H3BO4, 12 µm MnSO4, 1 µm ZnCl2, 1 µm CuSO4, 0.2 µm Na2MoO4) once a week for 4 weeks. To examine the effect of high temperature and drought, the nla and wild-type plants were grown in the full-nutrient soil LA4 (Sun Gro Horticulture). Eighteen days later, these plants were grown without watering for 8 days, or moved to a growth chamber with a temperature setting of 34°C for 3 h/25°C for 21 h for 10 days according to Yan et al. (2003). To investigate whether low nitrate application induces any phenotype in the roots of nla plants, both nla and wild-type seedlings were grown vertically in nitrogen-free medium (5.5 mm KH2PO4, 4.2 mm MgSO4, 2.5 mm CaCl2, 0.1 mm Fe-EDTA, 50 µm H3BO4, 12 µm MnSO4, 1 µm ZnCl2, 1 µm CuSO4, 0.2 µm Na2MoO4, 1% sucrose, 0.8% agar, pH 5.7) plates containing 0.5, 1 and 10 mm nitrate for 3 weeks.

Biochemical analysis

The fifth to eighth rosette leaves from nla and wild-type plants grown in LB2 soil with 3 mm nitrate for different lengths of time were harvested, frozen in liquid nitrogen and stored at −80°C for the following biochemical analyses. Nitrate contents in the frozen leaf powder were assayed as described by Cataldo et al. (1975). Total amino acids were extracted successively with 80%, 50%, 0% ethanol in HEPES–KOH buffer (pH 7.4), and the pooled supernatants were used for the total amino acid assay according to Rosen (1957). Soluble proteins were extracted from the frozen leaf power with 100 mm HEPES–KOH (pH 7.5) + 0.1% Triton X-100 and assayed using a commercial protein assay kit (Bio-Rad, http://www.bio-rad.com/). The percentages of total nitrogen in 1.5 mg dry leaf powder were measured by the Micro-Dumas combustion analysis method using the NA1500 C/H/N analyser (Carlo Erba Strumentazione; http://www.labinstruments.it/partn.html). Soluble sugar was extracted as described by Geiger et al. (1998), and assayed for glucose, fructose and sucrose contents using a commercially available kit (Megazyme, http://www.megazyme.com/). Starch was extracted from rosette leaves according to Delatte et al. (2005), and measured using a Megazyme total starch assay kit. Chlorophyll and anthocyanin in the frozen leaf powder were extracted and assayed according to Arnon (1949) and Noh and Spalding (1998), respectively.

Expression analysis by RT-PCR

Total RNA was extracted from various Arabidopsis plant tissues using TRIzol reagent (Invitrogen, http://www.invitrogen.com/). The first-strand cDNA was synthesized from total RNA samples with a kit (Fermentas, http://www.fermentas.com/) and used for PCR analysis. The expression of Arabidopsis genes RBCS, CAB1, SAG12 was detected by semi-quantitative RT-PCR, and ubiquitin-10 expression was used as the internal control. The specific primers and PCR conditions for these genes are available upon request.

Map-based cloning of the NLA gene, genetic complementation and determination of the nla mutant phenotype

One homozygous nla plant (in Columbia background) was crossed with a Landsberg erecta wild-type plant. Among the segregating F2 progeny, which was grown in LB2 soil with 3 mm KNO3, 518 plants showing the nla mutant phenotype were selected for PCR-based mapping. First round mapping was performed according to Lukowitz et al. (2000). The fine mapping was done by using SSLP and CAPS markers developed from the Arabidopsis genome sequence database (http://www.arabidopsis.org/), and these identified At1g02860 as one of the candidates for the NLA gene.

To determine whether At1g02860 is the NLA gene, the coding sequence of At1g02860 was amplified by RT-PCR using one pair of primers NLAcDNA-F (5′-ACAACCGGTTTGAGGGCTGAATTTGTTTG-3′) and NLAcDNA-R (5′-ACAGAATTCTATATCATATTCCAGTGAAGCT-3′). The PCR product was cloned into the binary vector pEGAD (Cutler et al., 2000). The construct was transformed into nla mutant plants as described by Clough and Bent (1998), and the transformants were screened by spreading the T1 seedlings with the herbicide BASTA (1:500 dilution, Aventis, http://www.sanofi-aventis.com/). T2 seeds from three independent T1 transgenic nla plants were sown in LB2 soil with 3 mm nitrate for phenotype testing.

Preparing transgenic plants expressing NLA promoter–GUS construct and histochemical GUS staining

A 1.5 kb fragment from the 5′ untranslated region of the NLA gene was amplified from Arabidopsis genomic DNA using the primers NLA-PROM-F (5′-ACAT CTAGAAACCTAGAACACTAGCAAGATG-3′) and NLA-PROM-R (5′-ACACCATGGACCATCAACAAACAAATTCAGC-3′). The PCR product was cloned into the GUS reporter gene fusion vector pCAMBIA3301 (CAMBIA, Canberra, Australia). The NLA promoter::GUS construct was transformed into Arabidopsis plants and transformants were screened with the herbicide BASTA as described above. The histochemical detection of GUS activity was performed according to Jefferson (1987).

Yeast two-hybrid analysis

Yeast two-hybrid analysis was performed using the BD Matchmaker Library Construction and Screening Kit (BD Biosciences, http://www.bdbiosciences.com/) according to the manufacturer’s protocol. The NLA and nla cDNAs were fused to the GAL4-binding domain of the bait vector pGBKT7. The cDNA library for the yeast two-hybrid analysis was generated with the total RNA from 18-DAG rosette leaves using the Matchmaker Kit, and co-transformed with linear pGADT7-Rec (containing the GAL4-activating domain) into the AH109 cell harbouring pGBKT7–NLA. The transformants were screened on the selection SD medium containing 2 mm 3-amino-1,2,4-triazole but lacking tryptophan, leucine and histidine. AtUBC8 was isolated as a positive clone, and thus pGADT7–AtBUC8 was re-transformed into the AH109 cells containing either pGBKT7–NLA, pGBKT7-nla or the control vector pGBKT7-53. The AH109 cells co-transformed with pGADT7–SV40 and pGBKT7-53 were used as the positive control.

Determination of the subcellular localization of GFP fusion proteins and the interaction between NLA and AtUBC8 in onion epidermal cells

To determine the subcellular localization of NLA, nla, RINGp and AtUBC8, the coding sequences for these proteins were amplified by PCR (the primers are available upon request), and cloned into the GFP reporter gene fusion vector pCAMBIA1302 (CAMBIA, http://www.cambia.org/). These constructs, including NLA:GFP, nla:GFP, RINGp:GFP and AtUBC8:GFP as well as the empty vector pCAMBIA1302 were transformed into onion epidermal cells and the fluorescent images were acquired as described by McCartney et al. (2004).

The bimolecular fluorescence complementation (Hu et al., 2002) was used to confirm the interaction between NLA and AtUBC8. The coding sequences for NLA, nla and RINGp proteins were fused to the N-terminus of the yellow fluorescence protein (YFP) in the vector E3082. AtUBC8 cDNA was fused to the C-terminus of YPF in the vector E3085. The construct pairs, E3085–AtUBC8 + E3082–NLA, E3085–AtUBC8 + E3082–nla, and E3085–AtUBC8 + E3082–RINGp, were co-transformed into onion epidermal cells and the fluorescence images of the cells were captured according to McCartney et al. (2004).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Dr Yuhai Cui (Agriculture and Agri-Food Canada, London, Ontario) for his advice on map-based gene cloning, Drs Stanton Gelvin and Lan-Ying Lee (Purdue University, USA) for the bimolecular fluorescence complementation system and Dr Joseph Colasanti and Dr Robert Mullen (University of Guelph, Canada) for a critical reading of the manuscript. We thank Mr Graham Smith and Ms Yeen Ting Huang for help with the onion epidermal cell transformation and Ms L. Hao and R. Zhao for technical help. This work is funded by the Natural Sciences and Engineering Research Council of Canada, Syngenta Inc., and the Ontario Research and Development Challenge Fund to SR.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. The effect of various nitrate applications on the growth and development of Arabiopsis plants (Columbia, Col) at 18 (a), 22 (b) and 26 (c) days after germination (DAG). Figure S2.NLA expression in Arabidopsis plants was analyzed by RT-PCR (a) and the NLA promoter-GUS transcription (b to f). GUS activity was visualized histochemically in various tissues, including (b) seedling; (c) root; (d) rosette leaf; (e) cauline leaf and stem; (f) flower; (g) developing siliques.

FilenameFormatSizeDescription
TPJ_3050_sm_FigS1.pdf3033KSupporting info item
TPJ_3050_sm_FigS2.pdf1670KSupporting info item

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