Proteinogenic l-amino acids (l-AAs) are essential in all kingdoms as building blocks of proteins. Their d-enantiomers are also known to fulfill important functions in microbes, fungi, and animals, but information about these molecules in plants is still sparse. Previously, it was shown that d-amino acids (d-AAs) are taken up and utilized by plants, but their ways to reduce excessive amounts of them still remained unclear. Analyses of plant d-AA content after d-Ala and d-Glu feeding opened the question if exudation of d-AAs into the rhizosphere takes place and plays a role in the reduction of d-AA content in plants. The exudation of d-Ala and d-Glu could be confirmed by amino acid analyses of growth media from plants treated with these d-AAs. Further tests revealed that other d-AAs were also secreted. Nevertheless, treatments with d-Ala and d-Glu showed that plants are still able to reduce their contents within the plant without exudation. Further exudation experiments with transport inhibitors revealed that d-AA root exudation is rather passive and comparable to the secretion of l-AAs. Altogether, these observations argued against a dominant role of exudation in the regulation of plant d-AA content, but may influence the composition of the rhizosphere.

D-Enantiomers of proteinogenic amino acids (D-AAs) are found ubiquitously, but the knowledge about their metabolism and functions in plants is scarce. A long forgotten phenomenon in this regard is the D-AA-stimulated ethylene production in plants. As a starting point to investigate this effect, the Arabidopsis accession Landsberg erecta (Ler) got into focus as it was found defective in metabolizing D-AAs. Combining genetics and molecular biology of T-DNA insertion lines and natural variants together with biochemical and physiological approaches, we could identify AtDAT1 as a major D-AA transaminase in Arabidopsis. Atdat1 loss-of-function mutants and Arabidopsis accessions with defective AtDAT1 alleles were unable to produce the metabolites of D-Met, D-Ala, D-Glu, and L-Met. This result corroborates the biochemical characterization, which showed highest activity of AtDAT1 using D-Met as a substrate. Germination of seedlings in light and dark led to enhanced growth inhibition of atdat1 mutants on D-Met. Ethylene measurements revealed an increased D-AA stimulated ethylene production in these mutants. According to initial working models of this phenomenon, D-Met is preferentially malonylated instead of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This decrease of ACC degradation should then lead to the increase of ethylene production. We could observe a reciprocal relation of malonylated methionine and ACC upon D-Met application and significantly more malonyl-methionine in atdat1 mutants. Unexpectedly, the malonyl-ACC levels did not differ between mutants and wild type. With AtDAT1, the first central enzyme of plant D-AA metabolism was characterized biochemically and physiologically. The specific effects of D-Met on ACC metabolism, ethylene production, and plant development of dat1 mutants unraveled the impact of AtDAT1 on these processes; however, they are not in full accordance to previous working models. Instead, our results imply the influence of additional factors or processes on D-AA-stimulated ethylene production, which await to be uncovered.

Keywords: D-amino acids in plants, D-amino acid-stimulated ethylene production, D-amino acid 9 specific transaminase, D-methionine, 1-aminocyclopropane-1-carboxylic acid, ethylene, amino acid 10 malonylation 11 Manuscript type: Original research 12Abstract 15 D-enantiomers of proteinogenic amino acids (D-AAs) are found ubiquitously, but the knowledge 16 about their metabolism and functions in plants is scarce. A long forgotten phenomenon in this regard 17 is the D-AA-stimulated ethylene production in plants. As a starting point to investigate this effect the 18Arabidopsis accession Landsberg erecta (Ler) got into focus as it was found defective in 19 metabolizing D-AAs. Combining genetics and molecular biology of T-DNA lines and natural 20 variants together with biochemical and physiological approaches we could identify AtDAT1 as a 21 major D-AA transaminase in Arabidopsis. Atdat1 loss-of-function mutants and Arabidopsis 22 accessions with defective AtDAT1 alleles were not able to produce D-Ala, D-Glu and L-Met, the 23 metabolites of D-Met, anymore. This result corroborates the biochemical characterization of 24AtDAT1, which showed highest activity using D-Met as substrate. Germination of seedlings in light 25and dark led to enhanced growth inhibition of atdat1 mutants on D-Met. Ethylene measurements 26revealed an enhanced D-AA stimulated ethylene production in these mutants. According to initial 27 working models of this phenomenon D-Met is preferentially malonylated instead of the ethylene 28 precursor 1-aminocyclopropane-1-carboxylic acid (ACC). This decrease of ACC degradation should 29 then lead to the increase of ethylene production. We could observe in our studies a reciprocal relation 30 of malonylated methionine and ACC upon D-Met application and even significantly more malonyl-31 methionine in atdat1 mutants. Unexpectedly, the malonyl-ACC levels did not differ between mutants 32 and wild type in these experiments. With AtDAT1, the first central enzyme of plant D-AA 33 metabolism was characterized biochemically and physiologically. The specific effects of D-Met on 34 ACC metabolization, ethylene production and plant development of dat1 mutants unraveled the 35 impact of AtDAT1 on these processes, but they are not in full accordance to previous working 36 AtDAT1 regulates ethylene production 2 models. Instead, our results imply the influence of additional candidate factors or processes on D-37 AA-stimulated ethylene production which await to be uncovered. 38

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