A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

Pyrylium based derivatization imaging mass spectrometer revealed the localization of L-DOPA.

Simultaneous imaging of l-dihydroxyphenylalanine (l-DOPA), dopamine (DA) and norepinephrine (NE) in the catecholamine metabolic pathway is particularly useful because l-DOPA is a neurophysiologically important metabolic intermediate. In this study, we found that 2,4,6-trimethylpyrillium tetrafluoroborate (TMPy) can selectively and efficiently react with target catecholamine molecules. Specifically, simultaneous visualization of DA and NE as metabolites of l-DOPA with high steric hinderance was achieved by derivatized-imaging mass spectrometry (IMS). Interestingly, l-DOPA showed strong localization in the brainstem, in contrast to the pattern of DA and NE, which co-localized with tyrosine hydroxylase (TH). In addition, to identify whether the detected molecules were endogenous or exogenous l-DOPA, mice were injected with l-DOPA deuterated in three positions (D3-l-DOPA), which was identifiable by a mass shift of 3Da. TMPy-labeled l-DOPA, DA and NE were detected at m/z 302.1, 258.1 and 274.1, while their D3 versions were detected at 305.0, 261.1 and 277.1 in mouse brain, respectively. l-DOPA and D3-l-DOPA were localized in the BS. DA and NE, and D3-DA and D3-NE, all of which are metabolites of L-DOPA and D3-l-DOPA, were localized in the striatum (STR) and locus coeruleus (LC). These findings suggest a mechanism in the brainstem that allows l-DOPA to accumulate without being metabolized to monoamines downstream of the metabolic pathway.

Optimization of the use of Py-Tag for next generation derivatization reagents in imaging mass spectrometry.

To demonstrate the accurate analysis of catecholamines and amino acid using derivatization reagents, we investigated the reaction conditions for 2,4,6-triethyl-3,5-dimethyl pyrylium trifluoromethanesulfonate (Py-Tag), derivatization of the targets dopamine (DA) and γ-aminobutyric acid (GABA) on tissue sections, and constructed an optimized reaction compartment. Ten different Py-Tag reaction conditions with the targets were considered. The optimal condition for the Py-Tag reaction with the targets was identified as a 70% methanol with 5% trimethylamine (v/v) solution at 60 °C under homogenous conditions. To reproduce this reaction on tissue sections, we constructed a reaction compartment to maintain humidity levels and facilitate the derivatization reaction. Moreover, visualization of DA and GABA was archived by derivatized-imaging mass spectrometry. Brain sections of unilateral 6-OHDA lesioned Parkinson's disease model rats showed Py-Tag DA (m/z 328.3) in the unilateral striatum and Py-Tag GABA (m/z 278.3) in the cerebral cortex, striatum, hippocampus and hypothalamus. Using the Parkinson's disease model rat brain, images with left-right differences were obtained for the localization of DA and GABA. These findings indicate that it is important to consider the reaction conditions that allow high reaction efficiency between DA or GABA and Py-Tag as well as high quality imaging of sections.

Visualization of dietary docosahexaenoic acid in whole-body zebrafish using matrix-assisted laser desorption/ionization mass spectrometry imaging.

Zebrafish models have been developed for several studies involving lipid metabolism and lipid-related diseases. In the present study, the migration of dietary docosahexaenoic acid (DHA) in whole-body zebrafish was estimated by stable-isotope tracer and matrix-assisted laser desorption/ionization mass spectrometry imaging. Administration of 1-13C-2,2-D2-labeled DHA ((+3)DHA) ethyl ester to male zebrafish was conducted to evaluate its accumulation, migration, and distribution in the body. The (+3)DHA content in the body of zebrafish after administering (+3)DHA for 10 and 15 d was significantly higher than that in the control group. (+3)DHA was observed as a constituent of phosphatidylcholine (PC) in the intestine of zebrafish that were administered (+3)DHA for 5 and 10 d. (+3)DHA-containing PC tended to accumulate in the intestines of zebrafish administered (+3)DHA for 1 d, indicating that recombination of (+3)DHA from ethyl ester to PC occurs quickly at intestine. After administration for 15 d, (+3)DHA-containing PC accumulated in the intestine, liver, and muscle of whole-body zebrafish. In contrast, (+3)DHA-containing PC was not detected in the brain. These results showed that dietary DHA is initially constructed into PC as a structural component of intestinal cell membranes and gradually migrates into peripheral tissues such as muscle.

Ammonium regulates the development of pine roots through hormonal crosstalk and differential expression of transcription factors in the apex.

Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium-induced changes in the transcriptome of maritime pine (Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT-ROOT, and phytohormone-related transcripts, such as ACO, involved in the development of the root meristem. Nano-PALDI-MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long-term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA-CK-ET phytohormone crosstalk and transcriptional regulation.

Localization analysis of essential oils in perilla herb (Perilla frutescens var. crispa) using derivatized mass spectrometry imaging.

The localization of essential oils, including flavor components, in perilla herb (Perilla frutescens var. crispa) were visually determined using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging. The surface of a perilla leaf was peeled using a cyanoacrylate adhesion compound and contained oil glands that retained their morphology and chemical properties. We imaged the three essential oils perillaldehyde, ß-caryophyllene, and rosmarinic acid (RA). Perillaldehyde was derivatized using glycine to prevent evaporation and allow its detection and imaging while localized in oil glands. ß-caryophyllene also localized in the oil glands and not in the epidermis region. RA was detected throughout the leaf, including the oil glands. Quantitative data for the three essential oils were obtained by gas chromatography- or liquid chromatography-MS. The concentrations of perillaldehyde, ß-caryophyllene, and RA were 12.6 ± 0.62, 0.27 ± 0.02, and 0.16 ± 0.02 [mg/g] in the paste sample of perilla herb. Peeling using a cyanoacrylate adhesion compound, and derivatization of a target such as an aroma component have great potential for mass spectrometry imaging for multiple essential oils.

Localization Analysis of Multiple Vitamins in Dried Persimmon (Diospyros kaki) Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry Imaging.

The drying process used for persimmon fruit (Diospyros kaki) can alter the composition of nutrients, and especially vitamins. We visually determined whether the amounts of vitamin A1, vitamin B6 and vitamin C vary after drying persimmon fruit, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging. Drying altered the amount of moisture between the fruit interior and surface. Vitamin A1 is lipophilic and localized at the desiccated outer regions (pericarp) and not in the inner region (mesocarp and endocarp), and its concentration was increased 3.4 times in dried fruit compared with raw persimmon. Vitamin B1 and B6 are water-soluble and concentrated in the moist mesocarp. The vitamin C content of dried persimmon is decreased by drying in the sun. The drying process affected the localizations and amounts of all the vitamins. The observed opposite localization of vitamin A1 compared to B1 and B6 was due to vitamin A1 being lipophilic and B1 and B6 being water soluble. Multiplevitamin imaging using MALDI-MSI has great potential for enhancing commodity value and for visually investigating the effects of manufacturing processes.