AtDAT1 is a key enzyme of D-amino acid stimulated ethylene production inArabidopsis thaliana

Abstract

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

d-Amino Acids in Plants: Sources, Metabolism, and Functions

“…This led to the hypothesis that the formation of d -Ala and d -Glu is driven by a d -AA specific transaminase [ 10 , 15 ]. Recently, we confirmed this assumption by the characterization of a previously identified transaminase [ 40 ], which contributes to the metabolization of d -AAs in Arabidopsis [ 41 ]. We could show in this study that loss-of-function mutants of this gene, AtDAT1 , almost completely lose the ability to move the amino group of any given d -AA to either pyruvate or 2-oxoglutarate (2-OG), leading to d -Ala and d -Glu, respectively ( Table 1 ).…”

“…Further analyses revealed that AtDAT1 is responsible for the decrease of d -Met content by transamination in Arabidopsis plants. The loss of this gene leads to an increased malonylation of d -Met and a reciprocal decrease of malonyl-ACC, the major product of the inactivation of the precursor of ethylene, ACC (1-aminocyclopropane-1-carboxylic acid), as shown in Figure 2 [ 41 ]. In this case, a d -AA ( d -Met) modulates the production of a hormone, and when the activity of the central processing enzyme is lost, the high level of d -Met outcompetes ACC to be malonylated by the N-malonyl-transferase (NMT).…”

“…Furthermore, it would be interesting to elucidate whether d -Glu, which is another common intermediate of d -AA catabolism in plants [ 14 , 15 ], also integrates into chloroplast envelopes, as it is usually found in bacterial peptidoglycan [ 1 ]. Apart from d -Ala other d -AAs were also identified to be involved in the developmental processes in plants— d -Cys was shown to be the precursor of the gasotransmitter H 2 S [ 51 ], and d -Met is involved in the regulation of the cellular content of the gaseous hormone ethylene [ 41 ]. Activation of NMDA receptors by d -Ser in animals was reported for plants, as it was shown that pollen tube growth depends on AtGLR1 activity, which is regulated by d -Ser [ 63 ].…”

“… Major metabolic pathways of d -Met and ACC in Arabidopsis , according to [ 41 ]. Green and red colored arrows indicate the decrease and increase of both educts and their products, respectively, in the course of d -Met accumulation, as it happens in plants with loss of DAT1 activity.…”

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.…”