AtDAT1 Is a Key Enzyme of D-Amino Acid Stimulated Ethylene Production in Arabidopsis thaliana

Abstract

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.

Evolutionary origin and functional diversification of aminotransferases

“…This class also contains the non-AT enzyme 4-amino-4-deoxychorismate lyase that is involved with folate biosynthesis. Interestingly, Arabidopsis d -amino acid AT also exhibits 4-amino-4-deoxychorismate lyase activity ( 122 , 123 ).…”

“…Arabidopsis DAAT is the major AT involved in metabolism of d -amino acids, which stimulate ethylene production through unknown mechanisms ( 123 ). Arabidopsis DAAT prefers d -methionine among many other d -amino acid substrates that can be used and utilizes both pyruvate and, to a lesser extent, α-ketoglutarate as amino acceptors (forming d -alanine and d -glutamate, respectively) ( 123 , 316 ). Arabidopsis DAAT also functions as 4-amino-4-deoxychorismate lyase for the synthesis of a folate precursor, aminobenzoate ( 122 ).…”

The regulation of ethylene biosynthesis: a complex multilevel control circuitry

“…We believe that multiple specialised enzymes exist to process d ‐amino acids and ACC. This theory is supported by a recent observation in Arabidopsis, in which d ‐amino acid transaminase 1 (DAT1) was shown to have a specialised role in d ‐amino acid metabolism (Suarez et al ., 2019). DAT1 is able to transaminate d ‐methionine into d ‐alanine, d ‐glutamic acid and l ‐methionine.…”

“…DAT1 is able to transaminate d ‐methionine into d ‐alanine, d ‐glutamic acid and l ‐methionine. The dat1 loss of function mutant showed an increased d ‐amino‐acid‐stimulated ethylene production compared with wild‐type plants when fed d ‐methionine (Suarez et al ., 2019). The authors also showed that this increase in ethylene production is not caused by a reduced malonylation of ACC, as both malonylated d ‐methionine (malonyl‐ d ‐methionine) and ACC (MACC) levels were higher in dat1 mutants (Suarez et al ., 2019).…”

“…The dat1 loss of function mutant showed an increased d ‐amino‐acid‐stimulated ethylene production compared with wild‐type plants when fed d ‐methionine (Suarez et al ., 2019). The authors also showed that this increase in ethylene production is not caused by a reduced malonylation of ACC, as both malonylated d ‐methionine (malonyl‐ d ‐methionine) and ACC (MACC) levels were higher in dat1 mutants (Suarez et al ., 2019). In conclusion, it appears that there are specialised enzymes that metabolise d ‐amino acids and conjugate ACC, but also that there are still unknown biochemical links that could explain the d ‐amino acid stimulated ethylene production in plants.…”