Although plants are permanently exposed to d-amino acids (d-AAs) in the rhizosphere, these compounds were for a long time regarded as generally detrimental, due to their inhibitory effects on plant growth. Recent studies showed that this statement needs a critical revision. There were several reports of active uptake by and transport of d-AAs in plants, leading to the question whether these processes happened just as side reactions or even on purpose. The identification and characterization of various transporter proteins and enzymes in plants with considerable affinities or specificities for d-AAs also pointed in the direction of their targeted uptake and utilization. This attracted more interest, as d-AAs were shown to be involved in different physiological processes in plants. Especially, the recent characterization of d-AA stimulated ethylene production in Arabidopsis thaliana revealed for the first time a physiological function for a specific d-AA and its metabolizing enzyme in plants. This finding opened the question regarding the physiological or developmental contexts in which d-AA stimulated ethylene synthesis are involved in. This question and the ones about the transport characteristics of d-AAs, their metabolism, and their different physiological effects, are the focus of this review.

For a long time D-enantiomers of proteinogenic L-amino acids were assumed to be physiologically irrelevant for plants. But there is growing evidence that D-amino acids (D-AAs) also fulfil important physiological functions in these organisms. However, the knowledge about the metabolic fate of D-AAs in plants is still scarce and more information about it is needed. To close this gap we established an optimized protocol for the processing and analysis of D- and L-AAs from large numbers of Arabidopsis lines. This included the application of 18 different D-AAs to seedlings, the extraction of free amino acids from the samples and the determination of 16 L-AAs and their corresponding D-enantiomers. To validate our approach we searched for genetic accessions with aberrant amino acid metabolism. Therefore we applied D-AAs on 17 ecotypes of Arabidopsis thaliana and analysed their free amino acid contents. These analyses confirmed the suitability of the system for the analysis of large sets of plant samples with enhanced velocity and improved accuracy. Furthermore, the resulting data led to the definition of standard amino acid profiles in response to D-AAs of Arabidopsis seedlings. Within these analyses the ecotype Landsberg erecta was found with aberrant metabolic patterns like drastically reduced capabilities to convert different D-AAs to D-alanine and D-glutamate. The presented experimental setup and results of this study offer starting points to dissect the metabolic pathway of D-AAs in plants.

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.

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.

Plastids in plants are assumed to have evolved from cyanobacteria as they have maintained several bacterial features. Recently, peptidoglycans, as bacterial cell wall components, have been shown to exist in the envelopes of moss chloroplasts. Phylogenomic comparisons of bacterial and plant genomes have raised the question of whether such structures are also part of chloroplasts in angiosperms. To address this question, we visualized canonical amino acids of peptidoglycan around chloroplasts of Arabidopsis and Nicotiana via click chemistry and fluorescence microscopy. Additional detection by different peptidoglycan-binding proteins from bacteria and animals supported this observation. Further Arabidopsis experiments with D-cycloserine and AtMurE knock-out lines, both affecting putative peptidoglycan biosynthesis, revealed a central role of this pathway in plastid genesis and division. Taken together, these results indicate that peptidoglycans are integral parts of plastids in the whole plant lineage. Elucidating their biosynthesis and further roles in the function of these organelles is yet to be achieved.

The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants.