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Keywords:

  • alanine aminotransferase;
  • AlaAT;
  • nitrogen metabolism;
  • anaerobic stress

Summary

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

Alanine aminotransferase (AlaAT) catalyses the reversible transfer of an amino group from glutamate to pyruvate to form 2-oxoglutarate and alanine. The regulation of AlaAT in several plant species has been studied in response to low-oxygen stress, light and nitrogen application. In this study, induction of Arabidopsis AlaAT1 and AlaAT2 during hypoxia was observed at the transcriptional level, and an increase in enzyme activity was detected in hypoxically treated roots. In addition, the tissue-specific expression of AlaAT1 and AlaAT2 was analysed using promoter:GUS fusions. The GUS staining patterns indicated that both AlaAT genes are expressed predominantly in vascular tissues. We manipulated AlaAT expression to determine the relative importance of this enzyme in low-oxygen stress tolerance and nitrogen metabolism. This was done by analysing T-DNA mutants and over-expressing barley AlaAT in Arabidopsis. The AlaAT1 knockout mutant (alaat1-1) showed a dramatic reduction in AlaAT activity, suggesting that AlaAT1 is the major AlaAT isozyme in Arabidopsis. Over-expression of barley AlaAT significantly increased the AlaAT activity in the transgenic plants. These plants were analysed for metabolic changes over a period of hypoxic stress and during their subsequent recovery. The results showed that alaat1-1 plants accumulate more alanine than wild-type plants during the early phase of hypoxia, and the decline in accumulated alanine was delayed in the alaat1-1 line during the post-hypoxia recovery period. When alanine was supplied as the nitrogen source, alaat1-1 plants utilized alanine less efficiently than wild-type plants did. These results indicate that the primary role of AlaAT1 is to break down alanine when it is in excess. Therefore, AlaAT appears to be crucial for the rapid conversion of alanine to pyruvate during recovery from low-oxygen stress.


Introduction

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

Transient flooding often causes oxygen deprivation in the rhizosphere. Such oxygen limitation adversely affects plant performance, as plant roots require sufficient oxygen supply to attain their full functions. Plants respond to low-oxygen stress by activating the pathway of anaerobic metabolism. The de novo synthesis of approximately 20 anaerobic polypeptides (ANPs) (Sachs et al., 1980) triggers the metabolic shift from oxidative phosphorylation to anaerobic fermentation. These ANPs, including the enzymes of glycolysis and anaerobic fermentation pathways, allow a limited amount of ATP synthesis in an oxygen-independent manner, typically resulting in the production of ethanol and lactate (Sachs et al., 1996; Vartapetian and Jackson, 1997). The parallel expression of genes encoding enzymes involved in nitrogen assimilation and amino acid metabolism also indicates that diverse metabolic pathways operate under low-oxygen conditions (Dennis et al., 2000; Liao and Lin, 2001). The production and accumulation of certain amino acids is indeed another common response of plants subjected to low-oxygen stress (Reggiani and Bertani, 2003; Ricard et al., 1994; Streeter and Thompson, 1972).

Alanine aminotransferase (AlaAT; E.C. 2.6.1.2) converts pyruvate and glutamate to alanine and 2-oxoglutarate. In doing so, it is involved in both carbon and nitrogen metabolism in plants. Arabidopsis contains four AlaAT homologues. Two of these homologues encode GGAT1 and GGAT2, which have both AlaAT activity and glutamate:glyoxylate aminotransferase (GGAT) activity (Igarashi et al., 2003; Liepman and Olsen, 2003). The presence of a peroxisomal targeting signal in both GGAT1 and GGAT2 suggests that they are localized in the peroxisomes (Liepman and Olsen, 2003), and subcellular fractionation analysis has found GGAT activity to be localized exclusively within Arabidopsis peroxisomes (Igarashi et al., 2003; Liepman and Olsen, 2003). GGATs appear to play important roles in photorespiration and amino acid metabolism (Igarashi et al., 2003; Liepman and Olsen, 2003). The other two genes, AlaAT1 and AlaAT2, have not been characterized in detail. In silico predictions suggest that AlaAT2 is mitochondrially localized while AlaAT1 is cytosolic (Liepman and Olsen, 2003). Cytosolic localization of a GFP–AlaAT1 fusion protein was observed in Nicotiana tabacum BY-2 cells (Igarashi et al., 2003).

The induction of AlaAT during hypoxia has been reported for several plant species including Hordeum vulgare (barley) (Good and Crosby, 1989; Muench and Good, 1994), Zea mays (maize) (Muench et al., 1998) and Glycine max (soybean) (Sousa and Sodek, 2003). Recent microarray studies have also demonstrated up-regulation of AlaAT1 in Arabidopsis during hypoxia (Klok et al., 2002; Loreti et al., 2005). Furthermore, induction of AlaAT by other stresses has been observed; for example, AlaAT expression is regulated by light and nitrogen stress in the leaves of Panicum miliaceum (Son et al., 1991, 1992). AlaAT was also up-regulated during recovery from nitrogen stress in maize (Muench et al., 1998). It is not unusual for enzymes to be involved in more than one stress response. Alcohol dehydrogenase (ADH) is likewise induced by several stresses, including wounding, low-oxygen, cold, ABA and heat (Dolferus et al., 1994a).

AlaAT expression has been detected in various tissues and organs. In addition to AlaAT expression in roots and leaf tissues, which is observed in most plant species, AlaAT expression has been detected in the inner endosperm tissues of developing seeds of Oryza sativa (rice) (Kikuchi et al., 1999) and floral organs in Arabidopsis (Igarashi et al., 2003). The versatile expression profile of AlaAT suggests the involvement of this enzyme in various physiological processes throughout the life cycle of plants.

Under hypoxic conditions, alanine is produced in relatively large amounts and becomes one of the most abundant amino acids in plant roots (Fan et al., 1997; Good and Muench, 1993; Reggiani et al., 1985; Sousa and Sodek, 2003; Streeter and Thompson, 1972). However, the mechanism of alanine accumulation and its ameliorating effect on low-oxygen stress have not been elucidated. It has been suggested that AlaAT is the key enzyme responsible for hypoxic alanine production based on its induction profiles in response to low-oxygen stress (Good and Crosby, 1989; Muench and Good, 1994). However, Sousa and Sodek (2003) point out that most of the increase in AlaAT activity takes place after alanine production ceases, suggesting that the major role of AlaAT occurs during the recovery from low-oxygen stress. While the purpose of hypoxic alanine production remains unclear, evidence suggests that it is important in the survival and nitrogen metabolism of plants under hypoxic conditions. In the single-cell alga Selenastrum minutum, most of the external ammonium assimilated under hypoxic conditions was incorporated into alanine (Vanlerberghe et al., 1991). Thus, alanine production under hypoxia is a mechanism by which the alga increases its nitrogen pool in preparation for the return to normal oxygen conditions. Supporting these findings is the observation that the anaerobic tolerance of rice coleoptiles depends upon the assimilation of inorganic nitrogen into alanine and other amino acids (Fan et al., 1997). In the legume Medicago truncatula, an inhibitor of azaserine (GOGAT) that indirectly blocks alanine synthesis impairs germination and seedling development under anoxic conditions (Ricoult et al., 2005).

We are interested in studying the roles of hypoxically inducible genes to improve flooding tolerance in plants. We have previously demonstrated that increasing metabolic flux through the ethanol fermentation pathway improves low-oxygen stress tolerance in Arabidopsis (Ismond et al., 2003). In this study, we have over-expressed barley AlaAT to assess the potential benefits of altering AlaAT expression under low-oxygen conditions. We have also analysed an AlaAT1 T-DNA knockout mutant to characterize the role of this enzyme in hypoxic alanine production and plant survival during low-oxygen stress. Our results suggest that hypoxic alanine synthesis does not require AlaAT activity; but indicate that AlaAT activity contributes to the rapid conversion of accumulated alanine to pyruvate during the post-hypoxia recovery period. Moreover, we have looked at the tissue-specific expression of both AlaAT1 and AlaAT2 using promoter:GUS fusions. Here we describe our findings regarding the regulation and functional characterization of AlaAT in Arabidopsis.

Results

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

Arabidopsis AlaAT gene family and their expression profiles

The Arabidopsis AlaAT gene family consists of four AlaAT genes (Igarashi et al., 2003; Liepman and Olsen, 2003). These four genes are subdivided into two groups, genes encoding AlaAT (AlaAT1, At1g17290; AlaAT2, At1g72330) and those encoding GGAT (GGAT1, At1g23310; GGAT2, At1g70580). Arabidopsis AlaAT1 and AlaAT2 have high sequence identity to the previously characterized AlaAT genes of other plant species such as barley (Muench and Good, 1994), maize (Muench et al., 1998) and rice (Kikuchi et al., 1999). Igarashi et al. (2003) have analysed the expression profile of each member of the Arabidopsis AlaAT gene family using real-time RT-PCR and have shown expression of AlaAT1 in both leaves and roots, as well as strong expression of GGAT1 in leaves. To confirm similar gene expression profiles in our growth system (see Experimental procedures), we examined expression levels of each gene using total RNA isolated from 17-day-old seedlings. Strong expression of AlaAT1 was seen in both the roots and shoots, whereas weak expression of AlaAT2 was detected only in the shoots (Figure 1). The expression of GGAT1 was observed in shoot tissue but not in root tissue (Figure 1). These results indicate that AlaAT1 is the major gene expressed in roots among the four members of the Arabidopsis AlaAT gene family.

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Figure 1.  Semi-quantitative RT-PCR of the AlaAT gene family. Total RNA isolated from shoots and roots of 17-day-old plants were used. PCR was carried out with two- or fourfold diluted aliquots of the synthesized first-strand cDNA (lanes 1/2 and 1/4, respectively), as well as undiluted aliquots (lane 1).

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Expression of AlaAT genes is induced by low-oxygen stress

The hypoxic induction of Arabidopsis AlaAT1 has been shown previously in microarray studies (Klok et al., 2002; Liu et al., 2005; Loreti et al., 2005). These experiments demonstrated that low-oxygen stress rapidly induced AlaAT1 expression upon onset of the stress (Figure 2a). A dramatic induction of the well-characterized anaerobically induced gene, ADH1, was also observed in the microarray experiment (Figure 2a). We analysed the expression profiles of the AlaAT gene family in response to low-oxygen stress. To do this, we performed semi-quantitative RT-PCR on total RNA samples isolated from plants subjected to various lengths of hypoxic stress (Figure 2b). In the roots, induction of both AlaAT1 and AlaAT2 was observed after 2 h of hypoxia, and peaked at 8 h (>fourfold increase). In the shoots, this up-regulation occurred more rapidly and was obvious for both genes after 2 h of hypoxic stress treatment. These results demonstrate that both AlaAT genes are induced during hypoxia. For GGAT1, there was no detectable message in the root tissue; however, induction was observed in shoot tissue after 8 h of hypoxia. The induction of GGAT1 in shoot tissue contrasts with what was observed in the microarray experiments (Figure 2a). As the different analyses were conducted at similar time points (6 h vs. 8 h), the reason for the discrepancy between these results is unclear. Expression of GGAT2 was virtually undetectable at all time points in roots and shoots.

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Figure 2.  Hypoxic induction profiles of AlaAT genes. (a) Microarray experiments showing the induction of AlaAT1 and ADH1 in response to anoxia. The data are taken from the microarray experiment performed by Loreti et al. (2005). RNA samples for the microarray were extracted from anoxically treated (6 h) and untreated 4-day-old Arabidopsis seedlings. The expression levels of each gene were compared between the two treatments. n/a, not available. (b) Semi-quantitative RT-PCR showing the increased expressions of AlaAT transcripts in roots and shoots in response to hypoxic stress. Total RNA was extracted from plants that had been hypoxically treated for the indicated period. PCR was carried out with two- or fourfold diluted aliquots of the synthesized first-strand cDNA (lanes 1/2 and 1/4, respectively) as well as undiluted aliquots (lane 1).

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AlaAT genes are preferentially expressed in vascular tissues

Transgenic plants expressing the β-glucuronidase (GUS) reporter gene under the control of the AlaAT1 and AlaAT2 promoters were stained for GUS activity under both aerobic and hypoxic conditions. Under aerobic conditions, pAlaAT1:GUS transgenic plants showed strong GUS expression in the vascular tissues of both roots and shoots (Figure 3a,e). GUS staining in the roots was noticeably fainter in the sections closer to the tip of the roots, and was absent in root tips as well as in the lateral root primordia (Figure 3a,b). In the leaves, preferential staining of vascular tissues was observed, but staining was also seen throughout the young rosette leaves (Figure 3e). pAlaAT2:GUS transgenic plants showed similar expression patterns in the shoots, but the coloration was fainter (Figure 3k). In addition, cotyledons and the first pair of rosette leaves were not stained intensely (Figure 3k). In the roots, vascular GUS expression was limited to the upper portions of the primary root (data not shown). Typically, GUS expression was absent in most of the root system, including the lateral roots, the lateral root primordia and the root tips (Figure 3g,h). Non-transgenic plants showed no background GUS activity (data not shown).

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Figure 3.  Tissue-specific expression of AlaAT genes. Transgenic plants expressing GUS under the control of the AlaAT1 or AlaAT2 promoters were stained for GUS activity. Untreated 14-day-old plants stained for 2 h are shown unless otherwise indicated. (a,b) Root of transgenic pAlaAT1:GUS plant. (c) Root of hypoxically treated plant. (d) Root of 20-day-old plant stained for 14 h. (e) Shoot of transgenic pAlaAT1:GUS plant. (f) Leaves of 20-day-old plant stained for 14 h. (g,h) Root of transgenic pAlaAT2:GUS plant. (i) Root of hypoxically treated plant. (j) Root of 20-day-old plant stained for 14 h. (k) Shoot of transgenic pAlaAT2:GUS plant. (l) Leaves of 20-day-old plant stained for 14h. The bars in (a–d) and (g–j) are 0.1 mm; those in (e,f,k,l) are 1.0 mm.

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In hypoxically treated plants, similar GUS staining patterns to those of untreated plants were observed for both pAlaAT1:GUS and pAlaAT2:GUS transgenic plants (Figure 3c,i). To detect low-level GUS activity, GUS staining was also performed overnight (14 h duration). When stained for this longer time period, more intense vascular staining of the roots was apparent for pAlaAT1:GUS plants (Figure 3d), but it was still faint for pAlaAT2:GUS plants (Figure 3j). The root tips remained unstained for both types of transgenic plants. In the shoots, GUS expression occurred predominantly in the vascular tissues, but weak staining was also detected in non-vascular leaf tissue for both pAlaAT1:GUS and pAlaAT2:GUS plants. For better resolution, the leaves of 20-day-old plants stained overnight are shown (Figure 3f,l).

To test whether the promoter fragments used in this study were capable of expressing GUS in a similar way to the native promoters, we confirmed that the hypoxic induction patterns of GUS were similar to those of AlaAT1 or AlaAT2 (Figure S1). The results indicate that the expression patterns of GUS shown in Figure 3 reflect the tissue specificity of the two endogenous AlaAT genes.

Isolation of an alaat1-1 mutant and transgenic plants over-expressing a barley AlaAT gene

We screened Salk T-DNA insertion lines (Alonso et al., 2003) in search of knockout mutants for AlaAT1 and AlaAT2 (for the complete list of the Salk lines screened in this study, see Table S1). We identified one mutant line containing a T-DNA insertion in AlaAT1 between the 8th and 9th exons (Figure 4a). Left border sequences were found on both sides of the insertion junction, suggesting the insertion of inverted repeats of the T-DNA. RT-PCR analysis indicated the absence of normal AlaAT1 transcript in this line (Figure 4b), thus we designated the mutant line alaat1-1. An aberrant product was amplified when using total RNA isolated from the shoots of alaat1-1, and appeared to be derived from non-specific annealing of the primer to a T-DNA region, based on sequence results (data not shown). Western blot analysis confirmed the absence of AlaAT protein in the alaat1-1 line (Figure 4c). To confirm the number of loci containing T-DNA insertions in alaat1-1, the knockout mutant was crossed with non-transgenic plants. Segregation of the kanamycin resistance gene, NPTII, was followed by PCR in the F2 population as NPTII was silenced in the mutant line. NPTII segregated in a 3:1 ratio, and was detected only in plants containing the T-DNA insertion within AlaAT1 (data not shown), suggesting that the alaat1-1 line contained the T-DNA insertion in only one locus.

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Figure 4.  Characterization of alaat1-1 mutant and AlaAT-over-expressing lines. (a) The gene structure of AlaAT1 and the site of T-DNA insertion in the knockout mutant (alaat1-1). Exon regions of AlaAT1 are shown by shaded boxes. (b) RT-PCR analysis showing the absence of normal AlaAT1 transcript in alaat1-1. Total RNA was isolated from roots and shoots of alaat1-1 and WT plants. PCR was carried out using synthesized first-strand cDNA with primers indicated as black triangles in (a). (c) Western blot analysis of alaat1-1 and WT. Proteins extracted from shoots and roots of alaat1-1 and WT plants were used: 10 μg protein were loaded into each lane. (d) RT-PCR analysis showing the expression of barley AlaAT in OxAlaAT lines. Total RNA was isolated from roots and shoots of OxAlaAT and WT plants. PCR was carried out using synthesized first-strand cDNA (+RT) or DNase I-treated RNA samples (−RT) and gene-specific primers. (e) Western blot analysis of OxAlaAT lines and WT. Proteins extracted from shoots and roots were used. For barley root and WT, 10 μg proteins were used, whereas 2 μg proteins were used for the OxAlaAT lines. Lanes correspond to those shown in (d).

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For AlaAT2, the only T-DNA insertions found were in the promoter region of the gene (Table S1). These insertions did not affect the expression of AlaAT2 under either aerobic or hypoxic conditions (data not shown). Thus, no AlaAT2 mutants were identified.

To produce transgenic plants with higher levels of AlaAT activity, we over-expressed a barley AlaAT cDNA under the control of the CaMV 35S promoter. Twenty independent transgenic lines were carried through to the T3 and T4 generations. Segregation of NPTII indicated that, of the lines selected (OxAlaAT#10, #15, #18 and #20; prefix ‘Ox’ for over-expression) based on the preliminary enzyme activity assay, all except OxAlaAT#18 contained a single insertion of the transgene. The OxAlaAT#18 line appeared to have the transgene inserted in two loci (data not shown). We confirmed the presence of the barley AlaAT transcript by RT-PCR (Figure 4d) and the protein by Western blot analysis (Figure 4e). These results indicated that the transgene was actively transcribed and translated in the selected OxAlaAT lines. However, the expression in OxAlaAT#15 was not uniform between different tissue types despite using a constitutive promoter. This may indicate a tissue-specific position effect.

Manipulated AlaAT enzyme activity in alaat1-1 and OxAlaAT lines

We measured AlaAT enzyme activity in the alaat1-1 mutant and the OxAlaAT lines under both aerobic and hypoxic conditions (Figure 5). Under aerobic conditions, the enzyme activity in the roots of alaat1-1 plants was negligible, and that in the shoots was reduced to 20–30% of wild-type (WT) activity. Hypoxic induction of AlaAT activity was only seen in the roots of WT plants after 24 h of hypoxia (approximately 1.6-fold). The results indicate that the T-DNA insertion in the AlaAT1 gene results in a dramatic decrease in overall AlaAT activity. Furthermore, the absence of activity in the roots of the alaat1-1 mutant suggests that AlaAT1 is the isozyme responsible for nearly all AlaAT activity in the roots of Arabidopsis. The low-level AlaAT activity detectable in the shoots of alaat1-1 could be attributed to GGAT1, which has been shown to be responsible for some AlaAT activity (approximately 40%) in photosynthesizing tissues (Igarashi et al., 2003).

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Figure 5.  Alanine aminotransferase activity in the alaat1-1 mutant and AlaAT-over-expressing lines. Seventeen-day-old plants that were treated for hypoxia (5% O2 for 24 h) and untreated plants were used. AlaAT activity was measured spectrophotometrically using the enzyme extracts of roots and shoots. Results shown are the means of triplicate samples. The error bars represent SE. The experiments were repeated with similar results.

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The three selected OxAlaAT lines showed significant increases in AlaAT activity (Figure 5). Under aerobic conditions, approximately 3.5–7-fold increases in activity were seen in the roots of the OxAlaAT plants. AlaAT activity levels were similarly elevated in the shoots of the OxAlaAT lines (approximately 4.5–10-fold increases). When subjected to hypoxic stress, AlaAT activity in the roots increased approximately 1.5-fold in WT plants, whereas smaller inductions were observed in the roots of the OxAlaAT lines (<1.2-fold increase). In the shoots, hypoxic induction of AlaAT activity was not evident in WT plants, and activity decreased in the OxAlaAT lines under hypoxia, perhaps as a result of a hypoxically triggered reduction in CaMV 35S promoter activity.

Metabolic and physiological analysis of alaat1-1 and OxAlaAT lines

To investigate how the mutation in AlaAT1 and over-expression of barley AlaAT affect metabolic profiles, we analysed the levels of several metabolites in alaat1-1 and the OxAlaAT lines (Figure 6). For these assays, plants were grown for 17 days and then subjected to hypoxia for various lengths of time. After 24 h of hypoxic treatment, plants were returned to aerobic conditions for up to 48 h. Plant samples taken at various time points during the hypoxic treatment and during the recovery period were used for metabolite assays.

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Figure 6.  Metabolic profiles of the alaat1-1 mutant over the period of hypoxic treatment and subsequent recovery. (a) Changes in the metabolites during over the period of hypoxia and post-hypoxia. Plants were subjected to hypoxia (5% O2) and the metabolites were extracted from the plants at four time points during the stress treatment (0, 2, 8 and 24 h). After 24 h of treatment, hypoxically treated plants were returned to normoxic conditions, and the samples were taken at three time points (12, 24 and 48 h). (b) Delayed decline in alanine concentration in alaat1-1 during the post-hypoxia recovery period. Plants were first hypoxically treated for 24 h, and samples were taken at seven time points over the 48 h recovery period to analyse the alanine decrease. The level of each metabolite was enzymatically determined. Results shown are the means of triplicate samples. The error bars represent SE. *P < 0.05 and **P < 0.01 (Student's t-test) for comparison of alaat1-1 with WT. The experiments were repeated with similar results.

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For ethanol and lactate, alaat1-1 and the WT plants showed almost identical patterns of accumulation in the external medium during hypoxic stress (Figure 6a). Upon return to aerobic conditions, both metabolites disappeared from the media at the same rate in both alaat1-1 and WT. A rapid increase in alanine concentration under hypoxia was observed in both alaat1-1 and WT plants (Figure 6a). Typically, alanine increased at a faster rate in the roots than in the shoots. In the roots of alaat1-1, alanine increased more rapidly than in WT plants, and significantly higher levels of alanine, relative to WT levels, were found after 2 h of hypoxia (1.34-fold). alaat1-1 plants also contained higher levels of alanine after 24 h of hypoxia, but this difference was not statistically significant and could not be repeated in other independent experiments. Upon return to aerobic conditions, alanine concentrations gradually decreased. During the recovery period, the decrease in alanine occurred more slowly in alaat1-1 than in WT plants, and significantly higher concentrations of alanine (1.59-fold) remained in the roots of alaat1-1 at 24 h after return to normoxia. In the shoots, significantly higher levels of alanine (1.74-fold) were detected in alaat1-1 plants compared with WT plants at 24 h after return to normoxia. The delay in the decline of alanine concentrations in alaat1-1 plants was verified in separate experiments with additional time points over the 48 h recovery period (Figure 6b). The results showed that the decrease in alanine concentration upon return to normoxia did not start in alaat1-1 until after the first 6 h of recovery, indicating the delay in alanine catabolism. In this experiment, the alaat1-1 plants contained more alanine than WT at all times during the recovery period in both shoots and roots (Figure 6b).

OxAlaAT lines produced and accumulated ethanol and lactate in the external medium at similar levels to WT plants during hypoxia (Figure S2). The accumulated metabolites disappeared from the media during the post-hypoxic period. Alanine concentrations increased at a similar rate in the OxAlaAT lines and WT plants during hypoxic treatment (Figure S2). During the recovery, one transgenic line, OxAlaAT#10, displayed a more rapid decrease of alanine in the roots; however, this was not observed in the other OxAlaAT lines. The level of glutamate was generally unaffected throughout the time course of the experiment in both OxAlaAT lines and WT plants, although the OxAlaAT lines contained more glutamate during the recovery period.

To evaluate low-oxygen stress tolerance in alaat1-1 and the OxAlaAT lines, we subjected the plants to either anoxic (100% N2) or hypoxic conditions (0.1% oxygen, 99.9% N2) for various lengths of time, and then scored the survival of the primary root tip based on its ability to maintain growth when returned to normoxic conditions. The decline in viability of the root tips over the period of anoxic stress was similar in alaat1-1 and WT plants (Figure S3). The root tips of OxAlaAT lines also behaved in a similar way to the WT plants (Figure S3). Hypoxic stress treatment yielded similar results. The shoot and root weights of plants that recovered from anoxic or hypoxic conditions were not significantly different between alaat1-1 and WT plants or between OxAlaAT lines and WT plants (data not shown).

The ability to utilize externally supplied alanine is affected in the alaat1-1 mutant

As the metabolite assays suggested that AlaAT generally converts alanine to pyruvate in vivo, we hypothesized that the alaat1-1 knockout would not be able to utilize alanine as a nitrogen source as effectively as WT plants. To test this hypothesis, plants were grown on MS medium containing various concentrations of inorganic nitrogen (KNO3:NH4NO3 = 1:1) with or without supplementation of alanine. When grown without alanine supplementation, no differences were observed between alaat1-1 and WT plants; however, when grown on low-nitrogen medium with alanine supplementation, alaat1-1 plants showed reduced growth compared to WT plants (Figure 7, Table 1). On alanine-supplemented media, WT plants grew approximately 1.3–1.5-fold larger than alaat1-1 plants. It should be noted, however, that the alaat1-1 line was still capable of utilizing the supplied alanine as indicated by the improved growth of alaat1-1 plants on low-nitrogen medium when supplemented with alanine. The OxAlaAT lines did not show any differences relative to WT plants when grown on these media. The results from a representative line (OxAlaAT#20) are shown in Table 1.

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Figure 7.  Reduced growth of the alaat1-1 mutant on alanine supplementation media. Plants were grown for 2 weeks on nitrogen-controlled 0.5 x MS medium with or without 5 mm alanine supplementation.

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Table 1.   Fresh weights (FW) of plants grown on alanine supplementation media FW (mg) of twenty 2-week-old plants grown on nitrogen-controlled 0.5 x MS medium with or without supplementation of 5 mm alanine were measured. Values shown are the means of triplicate plates ± SE
 Alanine concentration (mm)0 mm nitrogen0.4 mm nitrogen2 mm nitrogen10 mm nitrogen
  1. **P < 0.01 (Student's t-test) for comparison of alaat1-1 with WT. The experiments were repeated with similar results.

Col (WT)04.0 ± 0.225.3 ± 0.687.4 ± 5.9237.7 ± 9.8
alaat1-14.0 ± 0.825.0 ± 0.782.5 ± 2.0232.3 ± 11.9
Col (WT)584.6 ± 0.8133.1 ± 2.1177.0 ± 2.7245.9 ± 2.8
alaat1-1** 57.6 ± 2.0** 99.4 ± 4.3**134.2 ± 3.7225.3 ± 11.6
C24 (WT)03.3 ± 0.326.6 ± 0.295.1 ± 6.5175.0 ± 15.4
OxAlaAT#203.9 ± 0.126.2 ± 1.287.6 ± 4.7198.2 ± 11.8
C24 (WT)581.3 ± 1.997.9 ± 4.8129.8 ± 3.3215.6 ± 13.1
OxAlaAT#2074.9 ± 2.3101.2 ± 4.5141.1 ± 6.3212.4 ± 2.6

Discussion

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

The hypoxic induction of AlaAT is a well-known response of plants subjected to low-oxygen stress. The varied physiological roles of AlaAT are suggested by the diverse induction profiles of the genes by several different stresses and by the presence of multiple isoforms in distinct subcellular compartments, including the cytosol, mitochondria and peroxisome (Biekmann and Feierabend, 1982; Liepman and Olsen, 2004). In Arabidopsis, four AlaAT isozymes, each encoded by a member of the AlaAT gene family, have been described previously (Igarashi et al., 2003; Liepman and Olsen, 2003). In this study, we looked at the regulation and expression of AlaAT1 and AlaAT2, and attempted to elucidate the role of AlaAT through functional analysis of an alaat1-1 and transgenic lines over-expressing barley AlaAT in Arabidopsis.

The transcriptional induction of AlaAT genes in response to low-oxygen conditions has been demonstrated in barley (Muench and Good, 1994), maize (Muench et al., 1998) and M. truncatula (Ricoult et al., 2005). In Arabidopsis, several microarray experiments have demonstrated up-regulation of AlaAT1 in response to hypoxic stress; however, the response of AlaAT2 to hypoxia remained uncertain (Klok et al., 2002; Liu et al., 2005; Loreti et al., 2005). In one experiment, slight up-regulation of AlaAT2 was detected by microarray analysis, but this could not be repeated with real-time RT-PCR (Liu et al., 2005). In another study, the probes corresponding to AlaAT2 were not present on the Affymetrix ATH1 chip used in the experiment (Loreti et al., 2005). In the current study, we showed hypoxic induction of both AlaAT1 and AlaAT2 (Figure 2b). Our results demonstrate that transcript levels of both AlaAT1 and AlaAT2 increase quickly, plateau for a short period of time, and then slowly decrease. These induction profiles are similar to that of ADH1 in Arabidopsis (Dolferus et al., 1994a; Kürsteiner et al., 2003). However, as with ADH1, the degree of transcriptional induction of the AlaAT genes was significantly higher than the concomitant increase in enzyme activity (Figures 2b and 5). In general, the increase in enzyme activity required relatively longer stress treatments (>24–48 h). The slight increase of AlaAT activity after 24 h of hypoxia (Figure 5) is consistent with the increases in enzyme activity observed after 24 h of hypoxic treatment in barley (Good and Crosby, 1989), maize (Muench et al., 1998) and soybean (Sousa and Sodek, 2003). Moreover, the increase in AlaAT activity was limited to the roots, perhaps suggesting the inefficient translation of both AlaAT transcripts in the shoots under low-oxygen conditions.

The tissue specificity of both AlaAT transcripts was analysed by developing Arabidopsis transgenic plants expressing GUS under the control of either the AlaAT1 or AlaAT2 promoter. Analysis of the transgenic plants demonstrated that AlaAT1 is expressed at higher levels in the vascular tissues of both shoots and roots, while AlaAT2 is expressed predominantly in shoot vascular tissues. Similarly, AlaAT expression within vascular tissues has been shown in rice using GUS transcriptional fusions and in situ hybridization (Kikuchi et al., 1999). In this study, the absence of expression of both Arabidopsis AlaAT transcripts within root tips is noteworthy when compared to the expression of rice AlaAT in the root tip (Kikuchi et al., 1999) and that of other genes encoding ANPs, such as Arabidopsis ADH1 (Dolferus et al., 1994b). Furthermore, high expression of AlaAT1 also occurs in pollen as shown in the AtGenExpress expression atlas (Schmid et al., 2005). Pollen expression is also known for other ANPs, including pyruvate decarboxylase (PDC) and ADH, indicating the operation of an anaerobic-like metabolism in germinating pollen (Gass et al., 2005). Hypoxic stress did not affect the GUS staining patterns, although apparent transcriptional induction of both AlaAT and GUS driven by either of the AlaAT promoters were confirmed by semi-quantitative RT-PCR (Figure S1). This inconsistency may be attributed to the inefficient translation of GUS mRNA, which could be due to the absence of cis-elements from the GUS transcript that are required for translation under low-oxygen conditions. Alternatively, this could result from the fact that GUS staining occurs in the same tissues under hypoxic and normoxic conditions, and any increase in staining intensity would be minor, as is the case for the increased AlaAT enzyme activity.

Changes in metabolite levels during periods of hypoxia or anoxia have been closely monitored in a number of previous studies. A pronounced increase in alanine has been well documented in plants subjected to low-oxygen stress (Fan et al., 1997; Good and Muench, 1993; Sousa and Sodek, 2003; Streeter and Thompson, 1972). However, the metabolic changes upon return to aerobic conditions have not received the same attention. In this study, the products of anaerobic metabolism (ethanol, lactate and alanine) were measured during the post-hypoxic period. A steady decrease in alanine concentration was observed during the post-hypoxic period. Sousa and Sodek (2003) have also shown a similar but more rapid decrease in alanine concentration within 24 h post-hypoxia.

The involvement of AlaAT in hypoxic alanine production has long been speculated; however, compelling evidence has been lacking. We addressed this question by analysing the responses of alaat1-1 and the OxAlaAT lines to hypoxia. Under this stress condition, alaat1-1 plants accumulated high levels of alanine, and the increase in alanine content was even more rapid in alaat1-1 plants than in WT plants (Figure 6a). On the other hand, OxAlaAT lines accumulated alanine as rapidly as WT plants in response to low-oxygen stress (Figure S2). These results refute the hypothesis that the main role of AlaAT under hypoxia is the production of alanine. Instead, the delayed decline in alanine concentration in alaat1-1 plants upon return to normoxic conditions suggests that AlaAT activity is more important in the conversion of alanine during the post-hypoxic period (Figure 6a). Similarly, Sousa and Sodek (2003) concluded that the hypoxic induction of AlaAT is important to reduce elevated alanine levels to normal in a relatively short period of time upon return to normoxia. When subjected to hypoxic stress, plants are known to express genes whose products have functions during the subsequent recovery, ‘anticipating’ the return to normoxic conditions (Drew, 1997). The genes encoding enzymes responsible for the detoxification of reactive oxygen species generated during re-exposure to ambient oxygen (superoxide dismutase, peroxidase, ascorbate peroxidase, monodehydroascorbate reductase and glutathione reductase) are good examples (Geigenberger, 2003; Klok et al., 2002).

Manipulation of AlaAT activity did not alter the survival of the primary root tip under low-oxygen conditions. The root tips of alaat1-1 plants and the over-expressing lines showed similar responses to anoxic or hypoxic stress as those of the corresponding WT plants (Figure 7). The results suggest that AlaAT activity is not necessary for root tip survival under low-oxygen stress. The absence of GUS staining in the root tips (Figure 3) also supports a similar conclusion.

As alaat1-1 and OxAlaAT lines are perfectly viable and phenotypically normal, the physiological role of AlaAT remains ambiguous. However, the reduced growth of alaat1-1 plants on media supplemented with alanine suggests that AlaAT functions to convert excess alanine to pyruvate. This was also supported by the delayed decline of alanine in alaat1-1 plants during the post-hypoxic period. A similar conditional phenotype was observed in a mutant for cytosolic aspartate aminotransferase (aat2-2), where addition of aspartate (20 mm) to the media impaired root development (Miesak and Coruzzi, 2002). The inability of the aat2-2 mutant to negate the toxic effect of excess aspartate most likely caused the reduced growth of aat2-2 plants (Miesak and Coruzzi, 2002). In this study, we tested the impact of alanine supplementation on low-nitrogen media, thereby forcing the plants to utilize the amino N of alanine for growth. The inefficient utilization of the supplied alanine in the alaat1-1 line is the likely cause of the reduced growth of the alaat1-1 plants. The results also indicate that alanine was less efficient as a source of nitrogen, when compared to inorganic nitrogen. Indeed, 5 mm alanine resulted in growth that was equivalent to the effect of approximately 1.8–2.0 mm of inorganic nitrogen (Table 1). It has been demonstrated that amino acids can be an important form of organic nitrogen utilized by plants (Lipson and Näsholm, 2001; Thornton and Robinson, 2005). Our experiment demonstrates that Arabidopsis possesses the ability to uptake and utilize externally supplied alanine; however, the ability of plant roots to utilize amino acids under agricultural conditions remains uncertain.

Although the activity of AlaAT2 appears to be minor, the predicted mitochondrial localization (Liepman and Olsen, 2003) suggests that this isozyme is important in the targeted organelle. Under low-oxygen stress, the membrane structure of mitochondria is affected and mitochondria play a key role in the perception and signalling of falling oxygen levels within the cell (Subbaiah and Sachs, 2003; Vartapetian et al., 2003). Further characterization of the physiological role of AlaAT will require the identification and analysis of AlaAT2 mutants as well as a double knockout mutant of the two AlaAT genes, as the phenotype of alaat1-1 may have been diminished because of the redundant role of AlaAT2. Analysis of putative AlaAT2 mutants identified by TILLING (Colbert et al., 2001) is currently underway.

In summary, our results indicate that AlaAT1 primarily catalyses the conversion of alanine to pyruvate. The normal alanine level found in alaat1-1 plants under aerobic conditions suggests that Arabidopsis has multiple pathways for production and breakdown of alanine, and normal levels of alanine can be maintained without a functional AlaAT1 gene. Over-expression of AlaAT also did not alter alanine levels in the transgenic plants. This suggests that the AlaAT reaction is not the rate-limiting step for either alanine synthesis or breakdown. Given the vascular expression of both AlaAT transcripts, AlaAT activity may be important for the translocation of either alanine or pyruvate, and may facilitate maintenance of the carbon–nitrogen balance throughout 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 hypoxic stress treatment

Arabidopsis thaliana ecotypes Col-0 and C24 were used. Seeds were sterilized and sown on nylon mesh circles placed on top of 0.5 x MS medium containing 1% sucrose and 0.4% agar, unless otherwise indicated. Plants were grown under similar conditions as described by Ellis et al. (1999). Plants were grown on the nylon mesh plates for 10 days. Then, the plants grown on the nylon mesh were transferred from solid medium to liquid 0.5 x MS medium containing 1% sucrose, and were grown with gentle agitation on a rotary shaker for an additional 7 days. Tissues harvested from these 17-day-old plants were used for all RNA, protein and metabolite experiments. For the low-oxygen stress treatment, 16- or 17-day-old plants grown as described above were transferred to liquid 0.5 x MS medium containing 1% sucrose that had been first gassed for 15 min with 5% oxygen (balance nitrogen); 25 mg l−1 timentin was added to the liquid medium to prevent bacterial contamination. Four microfuge tubes cut in half, transversely, were placed inside the Petri dishes to slightly lift the lids and allow gas exchange. The plates were then stacked in anaerobic jars (Ismond et al., 2003). The anaerobic jars were flushed with 5% oxygen for an additional 15 min, and left in the dark for the duration of the treatments. Controls were performed by transferring the plants to aerobic liquid 0.5 x MS medium containing 25 mg l−1 timentin and placing on an orbital shaker in the dark for 24 h.

RT-PCR analysis

Total RNA was isolated from roots and shoots of 17-day-old plants using an RNeasy Plant Mini Kit (Qiagen; http://www.qiagen.com/). The isolated total RNA was treated with DNA-free DNase I (Ambion, http://www.ambion.com), and first-strand cDNA was synthesized using an oligo(dT)12−18 primer (Invitrogen; http://www.invitrogen.com/) and Superscript II reverse transcriptase (Invitrogen). PCR was performed using the synthesized first-strand cDNA and the following gene-specific primers: for AlaAT1, 5′-CTACGGTGAGAAGGATCTTG-3′ and 5′-TGCTCTCACATCTTAGCTTCTG-3′; for AlaAT2, 5′-GGGCTATGGAGAAAAAGATATCT-3′ and 5′-AGTTCATCTTCATGGTCAGGTC-3′; for GGAT1, see Liepman and Olsen (2003); for GGAT2, 5′-GAGTTCGGAAAGAAGTCGCTGAG-3′ and 5′-GAGTTTGACACAGAGTAGGACCAG-3′; for barley AlaAT, 5′-ACCTGATGCATTCTATGCTCTT-3′ and 5′-GGTTGTATGTAATCCCACATGTT-3′; for Actin2, 5′-CACTATGTTCTCAGGTATCGCT-3′ and 5′-TTTTAAGCCTTTGATCTTGAGAGC-3′; for β-Tubulin, see Kang and Singh (2000).

Analysis for tissue-specific expression of AlaAT transcripts

To generate promoter: GUS fusion constructs, approximately 1000 bp upstream regions of AlaAT1 and AlaAT2 were amplified from Col-0 genomic DNA using high-fidelity Platinum Taq DNA polymerase (Invitrogen) and cloned into pCR2.1-TOPO (Invitrogen) by TA-cloning using the following primer sets: for the AlaAT1 promoter, 5′-CCTCACTGCAAAACAGCAACA-3′ and 5′-TTGTGATTGTGTGGTATGGGA-3′; for the AlaAT2 promoter, 5′-GCTTCGAAGAACTATTAGTCCCAA-3′ and 5′-CTGTGAATCAGCTTTATACTAGGAAC-3′. The inserts were sequenced to confirm cloning of the correct promoter regions. To introduce EcoRI and NcoI sites at the ends of the promoter fragments, the cloned promoter regions were amplified by PCR using the following primers containing the desired restriction enzyme recognition sequences: EcoRI-AlaAT1, 5′-AACCGGAATTCCCTCACTGCAAAACAGC-3′; NcoI-AlaAT1, 5′-TCTTGACCATGGGTGATTGTGTGGTATGG-3′; EcoRI-AlaAT2, 5′-AACCGGAATTCGCTTCGAAGAACTATTA-3′; NcoI-AlaAT2, 5′-TCCTGACCATGGGTGAATCAGCTTTATACT-3′. The amplified products were cut with the corresponding restriction enzymes and inserted into pCAMBIA1305.1 (http://www.cambia.org/daisy/cambia/home.html). The constructs were then introduced into Arabidopsis plants (ecotype Col-0) using a modified Agrobacterium-mediated floral dip method (Clough and Bent, 1998). Transformed plants were selected based on hygromycin resistance, and a subset of T3 homozygous lines that displayed an observable GUS expression pattern were then selected.

The expression of both AlaAT promoters was examined at various developmental stages under aerobic conditions and after 24 h hypoxia. Plants were grown on vertical 0.5 x MS plates (1% sucrose, 1% agar) for 9–20 days. Plants were then hypoxically treated (5% oxygen) for 24 h in the dark, while control plants were left in air for 24 h in the dark. In vivo GUS staining was performed as described previously (De Block and Debrouwer, 1992), with slight modifications to allow the staining of whole Arabidopsis seedlings. The plants were incubated at 37°C in the staining buffer (50 mm NaHPO4, 2 mm potassium ferrocyanide, 2 mm potassium ferricynide, 0.2% Triton X-100 and 2 mm X-Gluc) for 2 h or overnight (14 h). A minimum of four independent transgenic lines were used for the histological studies. Untransformed plants were used as controls.

Development of AlaAT-over-expressing transgenic lines

For over-expression of AlaAT, the CaMV 35S promoter was used. The construct was created by introducing a barley AlaAT cDNA (Z26322) into the EcoRI/XbaI sites of plasmid pART7 (Gleave, 1992). The expression cartridge, consisting of the cDNA flanked by the CaMV 35S promoter and the octopine synthase gene (ocs) 3′ UTR, was transferred into the NotI site of the corresponding binary vector pART27. The construct was then introduced into Arabidopsis plants (ecotype C24) using a modified root transformation protocol (Dolferus et al., 1994b). Transformed plants were selected based on kanamycin resistance, and homozygous T3 or T4 seeds were used for all experiments.

Isolation of the AlaAT1 knockout line

The AlaAT1 knockout line was isolated from Salk T-DNA insertion lines (Alonso et al., 2003) obtained from the Arabidopsis Biological Resource Centre (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm), using the PCR screening scheme described at http://signal.salk.edu. Primers specific for the left border sequence of T-DNA (LBb1; 5′-GCGTGGACCGCTTGCTGCAACT-3′) and for AlaAT1 (5′-GCGTGCTTGGAAGATTCTGGA-3′ and 5′-GGAATGGAACTTTTTGTCAGGGA-3′) were used for the screening of plants homozygous for the T-DNA insertion. The primers for AlaAT1 were also used for RT-PCR analysis on the alaat1-1 line. For segregation analysis of the kanamycin resistance gene, primers for NPTII (5′-CGCTCAGAAGAACTCGTCAAGAA-3′ and 5′-TTTGTCAAGACCGACCTGTCC-3′) were used for PCR analysis.

Enzyme and metabolite assays

Plants that were hypoxically treated for 24 h or untreated plants were used. AlaAT enzyme extractions and activity measurements were conducted as previously described (Ismond et al., 2003). Harvested tissues were ground in an extraction buffer (5 mm EDTA, 1 mm DTT, 10 mm cysteine, 0.1 mm PMSF, 5 μm leupeptin and 100 mm Tris, pH 7.5) with a pinch of sand and PVPP, and then centrifuged for 15 min (16 000 g) to remove the cell debris. AlaAT activity was measured for the reverse reaction (alanine to pyruvate) in assay buffer (100 mm Tris, pH 8.0, 10 mm 2-oxoglutarate, 0.28 mm NADH, 70 mm alanine and 1.2 U ml−1 lactate dehydrogenase). Alanine was added last to initiate the reaction, and the concomitant decrease in absorbance at 340 nm by oxidation of NADH was monitored. Protein measurements for all extracts were performed using a Bio-Rad protein assay kit (http://www.bio-rad.com/) with BSA as a standard.

Aerobically (0 h treatment) and hypoxically treated plants were used for metabolite extractions. Samples were taken after 2, 8 or 24 h of hypoxic treatment. The stressed plants were subsequently returned to normoxic conditions after 24 h of hypoxic treatment, and samples were taken at 12, 24 and 48 h after the return to normoxia. Metabolite extractions were carried out as described previously (Good and Muench, 1993). Harvested tissues were flash-frozen in liquid nitrogen and immediately ground using a mortar and pestle in 0.6 m perchloric acid with a pinch of sand. The perchloric acid extracts were then neutralized with 3 m NaOH and stored at −70°C until used for the assay. Levels of pyruvate, lactate, ethanol, alanine and glutamate were measured by enzyme-linked assays using a 96-well ELISA plate reader (SpectraMax+) (Molecular Devices, Sunnyvale, CA, USA; http://www.moleculardevices.com) at room temperature (Ismond et al., 2003). Samples were spiked with known amounts of metabolites to test the accuracy of the assay. Triplicate samples were used for all experiments, and the experiments were repeated at least twice for each time point.

Western blot analysis

Soluble proteins were extracted from shoots and roots using enzyme extraction buffer (see above) and then separated on a SDS polyacrylamide gel (7.5% running gel/4% stacking gel) using a Mini-Protein II electrophoresis cell (Bio-Rad). Proteins were then transferred to nitrocellulose membrane using a Mini Trans-Blot Cell (Bio-Rad; http://www.bio-rad.com) according to the manufacturer's specifications. Primary antibody raised against purified barley AlaAT2 (Good and Muench, 1992; Muench and Good, 1994) was used. Antibody staining was performed using an ECL Western detection kit (Amersham Biosciences; http://www5.amershambiosciences.com/) with Western blocking reagent from Roche Applied Science (http://www.roche-applied-science.com) instead of non-fat dry milk to improve the specific binding of the primary antibody to the AlaAT proteins.

Root tip survival assay

To assess the anaerobic tolerance of the plants, a root tip survival assay was performed as described previously (Ellis et al., 1999) with slight modifications. Sterilized seeds were sown on nylon mesh strips overlying 0.5 x MS medium solidified with 1 % agar and supplemented with 1% sucrose. Plates were placed upright to allow growth of the roots on the surface of the medium only. After 7 days, the nylon mesh strips supporting the seedlings were transferred to liquid 0.5 x MS medium containing 1% sucrose and 25 mg l−1 timentin that had been previously gassed with full nitrogen gas or 0.1% oxygen gas (balanced with nitrogen) for 15 min. Microfuge tubes that had been cut in half, transversely, were placed inside the Petri dishes to slightly lift the lids and ensure rapid gas exchange. The plates were then stacked into anaerobic jars and flushed with full nitrogen gas or 0.1% oxygen gas for an additional 15 min. The anaerobic jars were kept in the dark for the duration of hypoxic treatment. At the end of the stress treatment, 15 plants per plate were randomly selected and transferred to square Petri dishes containing 0.5 x MS medium with 1% agar and allowed to recover for 1 week. Root tip survival, based on the ability of the primary root tip to re-grow, was scored 2 days after transferring seedlings to the new square plates. At the end of the recovery period, shoot and root survival, as well as shoot and root weights, were recorded. Triplicate samples were used in all experiments and the experiments were repeated three times.

Alanine supplementation test

To assess the ability of the plants to utilize alanine as nitrogen source, plants were grown on controlled 0.5 x MS medium (0.6% agar) with varying concentrations of inorganic nitrogen (0–10 mm inorganic nitrogen, KNO3:NH4NO3 = 1:1), with or without 5 mm alanine supplementation. Two sucrose concentrations, 3% and 0.3%, were also used. Twenty-two seeds of the OxAlaAT lines or the alaat1-1 mutant were co-plated with corresponding WT seeds on the same plates. After 2 weeks of growth, the plates were photographed and the fresh weight and dry weight of the above-ground tissues of 20 plants were measured. Triplicates were used in all experiments and the experiments were repeated three times.

Acknowledgements

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

We would like to thank Mark Wolansky and Mary DePauw for technical assistance. This work was supported in part by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to A.G.G. R.D. was financially supported by the Cooperative Research Centre for Sustainable Rice Production (c/o New South Wales Institute of Agriculture).

References

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

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. Semi-quantitative RT-PCR showing the hypoxic induction of GUS driven by AlaATs promoters. Total RNA was extracted from the transgenic plants that have been hypoxically treated for the indicated period. PCR was carried out with 2- or 4-fold diluted aliquots of the synthesized first strand cDNA (lane ½ and ¼) as well as undiluted aliquots (lane 1). Figure S2. Metabolic profiles of OxAlaAT lines over the period of hypoxic treatment and the subsequent recovery. Plants were subjected for hypoxia (5% O2) and the metabolites were extracted from the plants harvested at 4 time points during the stress treatment (0 h, 2 h, 8 h, and 24 h). After 24 h of treatment, hypoxically treated plants were returned to normoxic conditions, and the samples were taken at 3 time points (12 h, 24 h and 48 h). The level of each metabolite was enzymatically determined. Results shown are the means of triplicate samples. The error bars are S.E. * and ** indicates P<0.05 and P<0.01 (Student?s t-test), respectively. The experiments were repeated with similar results. Figure S3. Root tip survival under anoxic conditions. 7-day-old seedlings grown on vertical plates were subjected to various lengths of anoxic conditions (full N2, for 0 h-24 h) in liquid media. The stressed seedlings were then transferred to new vertical plates and allowed to recover. The root tip survival was scored based on the ability of the primary root tip to grow further at 2 days after the transfer. (a) Root tip survival rate of alaat1-1 and WT. (b) Root tip survival rate of OxAlaAT lines and WT. Results shown are the means of 3 replicate samples. The error bars are S.E. The experiments were repeated with similar results. Table S1. Summary of the screening of Salk T-DNA lines. The T-DNA insertions in each line were confirmed by PCR analysis. The actual insertion sites were confirmed by sequencing the gel purified PCR products. AlaAT activity was measured in shoots and roots of each line. 17-day-old seedlings grown as described in Experimental procedures were used for the enzyme activity assay. (n/a: not available, n.d.: not detected, )

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TPJ_3023_sm_FigureS1.zip1484KSupporting info item
TPJ_3023_sm_FiguresS2S3.zip367KSupporting info item
TPJ_3023_sm_TableS1.doc35KSupporting info item

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