In this study a versatile method was presented to analyse the contents of D- and L-AAs in a large number of plant samples. In a proof of principle experiment with 17 ecotypes of Arabidopsis thaliana general metabolic reactions of plants against D-AAs could be either verified or determined for the first time with this method. The major goal of this approach was the establishment of a technique to identify genetic lines with aberrant metabolism of D-AAs. The identification of Ler-0 as an ecotype with a lack to convert various D-AAs either to their corresponding L-AAs or to D-Ala and D-Glu can be accounted as a successful application of this method. Furthermore, the identification of an ecotype with aberrant D-AA metabolism provides first insights into its mechanisms.
A variety of methods and protocols have been published to detect and analyse D- and L-AAs from plant extracts (Ali et al. 2006; Brückner and Westhauser 1994
2003; Herrero et al. 2007). In a previous study (Gördes et al. 2011) a method was described, which represented an alternative to existing methods specifically developed for the analysis of Arabidopsis plant material. The described procedure in this study represents an improvement to this method. As it was shown above the portion of quantifiable data increased with the present method (Table 3). The improved range of quantitation might explain this effect. The upper LOQ for the present study was determined at 1,000 μmol/L, with a lower LOQ of 0.5 μmol/L (see Additional file 1: Table S2). In Gördes et al. (2011) the range of detection was between 1.25/5-500 μmol/L. This means, that an improvement of sensitivity from 5–20 fold was achieved by the optimised system described in the present study. Furthermore it has been shown in preliminary studies that this method is applicable to other plant species ranging from mosses over monocots to different dicots (data not shown). Although final validation of the latter is still pending this would point to a general applicability of this procedure for many different plant species.
Besides the extended concentration range additional important improvements of the described method are the reduction of the overall analysis time per sample and the exceptionally long-term stability. Compared to the chromatographic method in Gördes et al. (2011), the time was cut by half (12 min instead of 25 min). Due to this time reduction up to 120 measurements were possible within 24 hours. For this effect, an optimised mobile phase gradient, downsized column dimensions, and the implementation of an alternating column regeneration (ACR, Agilent Technology) mode were necessary. With ACR, a system of two identical columns allows the accomplishment of the complete cleaning and equilibration process in parallel to the chromatographic measurement. Furthermore, in combination with a reduced injection volume the column lifetime could be increased by factor of 10, up to several thousand measurements.
The major goal of the presented work was the analysis of different accessions of A. thaliana to identify ecotypes with aberrant responses against D-AAs. Therefore a large number of AA measurements were performed. A general observation over all measurements was that the vast majority of changes in all accessions in response to exogenous D-AAs represented increases of AA levels as only few decreases were found (Tables 4, 5, Additional file 1: Tables S3 and S4). This supports the hypothesis that plants acquire and metabolise D-AAs as nitrogen sources (Hill et al. 2011, Vranova 2012).
The large number of ecotypes, treatments and measurements may also have contributed to answer a question, which arose in Gördes et al. (2011). There it was asked why just D-His, D-Met, D-Phe and D-Trp led to putative racemisations. In the light of the actual results it can be said that D-Ile, D-Leu, D-Ser and D-Thr also cause a similar effect. The conversion rates are more moderate and do not appear as regularly as for the first four D-AAs (Table 5). But the putatively racemised D-AAs do not seem to be limited to D-His, D-Met, D-Phe and D-Trp. Currently it cannot be said if other D-AAs also could cause putative racemisations in other ecotypes. Furthermore, the problem remains that for most of these 8 D-AAs no racemase from plants is known. A plant alanine racemase had been characterised, but is still not identified (Ono et al. 2006 Nishimura et al. 2007). A serine racemase however has been isolated and characterised from different plant species. This enzyme possesses an additional L-serine dehydratase activity, which would possibly explain the moderate conversion rates (Fujitani et al. 2006,2007, Gogami et al. 2009).
One prerequisite to identify plant accessions with aberrant reactions against D-AAs was the observation and definition of common reactions in the analysed plants. As an outcome of this study three major classes of reactions in response to applied D-AAs were defined (see Results). It was interesting in this respect that each class of reactions was not just specified by the type of reaction but also by extent and distribution. In the first reaction class (D-AA → L-non-enantiomer) the rate of reaction at 1.5-5 fold was relatively small and in some ecotypes just one particular reaction of this class was observed, whereas in other ecotypes almost all of them appeared. The relatively low rate of changes and their facultative appearance in different ecotypes might be a reason why this reaction class was not reported in previous studies (Chen et al. 2010; Gördes et al. 2011). In comparison the reaction rates of the second class (D-AA → L-enantiomer) and the third class of reactions (D-AA → D-Ala/Glu) were much higher with increases up to >700 fold (D-Met → D-Ala; Additional file 1: Table S3). Only the conversion of D-AA → D-Ala could be observed in almost all ecotypes and after all treatments. Instead putative racemisations and D-AA → D-Glu conversions were found often, but not in such regular manner. Especially the high rates in the latter two reaction classes may be an indicator that these reactions are directly catalysed, whereas the observed conversions summarised in the first class of reaction are indirect changes in response to D-AA application and therefore of a rather secondary type.
The identification of the ecotype Landsberg erecta as an accession with drastically reduced abilities to metabolise exogenously applied D-AAs can be accounted as a proof of the presented concept for the investigation of plant D-AA metabolism. As one example there is still the unanswered question about the catalytic mechanisms leading to the formation of enantiomers and specific D-AAs. Vranova et al. (2012) suggested racemisation, deamination or transamination as putative reactions being responsible for the observed phenomena. The aberrant responses of Landsberg erecta may serve as a genetic starting point to answer the posed question.
All applied D-AAs were detectable in reasonable amounts in the analysed plant extracts (Additional file 2). Due to this fact, defective import of D-AAs, as observed in the AA transporter mutant lht1 (Gördes et al. 2011), can be excluded. Instead a defect in D-AA metabolism has to be assumed. In principle there are two possible genetic scenarios which would explain the results in Ler-0. Both scenarios are schematised in Figure 5. The assumption in the first model is that at least two enzymes are responsible for the effects in Ler-0 (Figure 5A). An unspecific AA racemase leads to the conversion of D-AAs to their corresponding enantiomers, and a D-AA aminotransferase is responsible for the evolution of D-Ala and D-Glu. This means that at least two genes are mutated in Ler-0. Furthermore, this model requires an unspecific AA racemase. But such a single enzyme has not been shown yet (for an overview about AA racemases see Conti et al. 2011). This would mean that probably more than one candidate racemase gene got lost in Ler-0 to explain the presented results. This makes this model less probable than the second one (Figure 5B), which proposes just the loss of function of a single enzyme, a D-AA aminotransferase of primary responsibility. According to this scenario the observed putative racemisation in almost all ecotypes would then be the result of secondary L-AA aminotransferase reactions using the remaining keto acid from the D-AA aminotransferase reaction with an available L-AA. There are several highly expressed L-AA aminotransferases in Arabidopsis cells which are responsible for a number of reactions in primary and secondary metabolism. A loss of such a primary D-AA aminotransferase function in Ler-0 would explain the results. The characterisation of a D-AA aminotransferase has been reported previously (Funakoshi et al. 2008) and it has been shown that such enzymes are expressed in Arabidopsis, but it is not known yet if this enzyme or putative functional homologs can act in the proposed way. Nevertheless, the aberrant reaction of Ler-0 points to a defect in D-AA aminotransferase reaction and makes this reaction also most probable as the primary cause for D-AA conversion in the list of candidates of Vranova et al. (2012).
Furthermore, the presented data raise the question about the physiological significance of D-AAs in plants. From previous studies it is known that not all D-AAs have detrimental effects on Arabidopsis plants and that some of them (e.g. D-Val, D-Lys, D-Ile) even promote plant growth (Erikson et al. 2004 Chen et al. 2010; Gördes et al. 2011). Recently it was shown that wheat plants are able to take up and utilise D-Ala (Hill et al. 2011). This was the first time it was shown that plants are capable to make use of D-AAs as a source of nitrogen supply. The results presented in this study revealed, beside the aberrant reaction of Ler-0, gradually differing amino acid conversions in the analysed ecotypes. These observations might reflect the varying capabilities of different accessions to utilise D-AAs. Another possibility for the different amino acid profiles in the ecotypes might be their different capacities to degrade D- and L-AAs leading to differential accumulation of amino acids.
Being a source of nitrogen may be one function of D-AAs in plants. As previously shown for other organisms like bacteria or mammals particular D-AAs like D-Ala, D-Glu or D-Ser also act as regulatory molecules (for overviews see Friedman 2010; Cava et al. 2011). In Arabidopsis such a relationship was recently shown for the first time by the influence of D-Ser on pollen tube development. Furthermore, it was revealed that D-serine racemase is involved in D-Ser mediated signal transduction (Michard et al. 2011). In this respect it would be interesting to compare pollination of the different accessions with their capability to metabolise D-Ser. Nevertheless, the role of D-Ser and possibly other D-AAs as regulatory molecules and the involvement of a D-AA metabolising enzyme in a D-AA influenced developmental process point out the importance of the homeostasis of D-AAs in plant life cycle.
The method presented in this report for amino acid profiling might contribute to addressing some of the questions given above due to its potential for the analysis of large numbers of plant samples. The processing and analysis of 17 Arabidopsis ecotypes confirmed the capabilities of the method. The wide variety of amino acid profiles in these accessions, especially the identification of Ler-0 as an ecotype with aberrant reactions against D-AAs, revealed a great plasticity of A. thaliana to metabolise these molecules. Further genetic and molecular analyses of D-AA metabolism of the given ecotypes, additionally of knock out mutants of candidate genes, but also of other plant species, should result in a larger insight into this metabolic pathway. Detailed comparison of amino acid profiles and morphological and physiological phenotyping of these plants in response to D-AAs should also answer the question if D-AAs have additional regulatory functions or act primarily as nitrogen sources.