Synthesis and spectroscopic characterization of derivatives of proteinogenic amino acids, simultaneously labelled with 13C, 15N and 2H in the backbone

The synthesis of small organic molecules, known as specialized or secondary metabolites, is one mechanism by which plants resist and tolerate biotic and abiotic stress. Many specialized metabolites are derived from the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr). In addition, the improved characterization of compounds derived from these amino acids could inform strategies for developing crops with greater resilience and improved traits for the biorefinery. Sorghum and other grasses possess phenylalanine ammonia-lyase (PAL) enzymes that generate cinnamic acid from Phe and bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) enzymes that generate cinnamic acid and p-coumaric acid from Phe and Tyr, respectively. Cinnamic acid can, in turn, be converted into p-coumaric acid by cinnamate 4-hydroxylase. Thus, Phe and Tyr are both precursors of common downstream products. Not all derivatives of Phe and Tyr are shared, however, and each can act as a precursor for unique metabolites. In this study, 13C isotopic-labeled precursors and the recently developed Precursor of Origin Determination in Untargeted Metabolomics (PODIUM) mass spectrometry (MS) analytical pipeline were used to identify over 600 MS features derived from Phe and Tyr in sorghum. These features comprised 20% of the MS signal collected by reverse-phase chromatography and detected through negative-ionization. Ninety percent of the labeled mass features were derived from both Phe and Tyr, although the proportional contribution of each precursor varied. In addition, the relative incorporation of Phe and Tyr varied between metabolites and tissues, suggesting the existence of multiple pools of p-coumaric acid that are fed by the two amino acids. Furthermore, Phe incorporation was greater for many known hydroxycinnamate esters and flavonoid glycosides. In contrast, mass features derived exclusively from Tyr were the most abundant in every tissue. The Phe- and Tyr-derived metabolite library was also utilized to retrospectively annotate soluble MS features in two brown midrib mutants (bmr6 and bmr12) identifying several MS features that change significantly in each mutant.

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Search for molecular biosignatures on Mars: laboratory simulations of UV irradiation of molecule-mineral complexes

10.5194/epsc2021-705 ◽

2021 ◽

Author(s):

Teresa Fornaro ◽

Giovanni Poggiali ◽

Maria Angela Corazzi ◽

Cristina Garcia ◽

Giulia Dimitri ◽

...

Keyword(s):

Amino Acids ◽

Organic Compounds ◽

Uv Irradiation ◽

Organic Molecules ◽

Spectroscopic Characterization ◽

Sample Return ◽

Laboratory Simulations ◽

Prebiotic Molecules

<div>&#160;</div> Abstract We present laboratory activities of preparation, characterization, and UV irradiation processing of Mars soil analogues, which are key to support both in situ exploration and sample return missions devoted to detection of molecular biosignatures on Mars. In detail we prepared analog mineral samples relevant to the landing sites of past, present and future Mars exploration missions, such as Gale Crater, Jezero Crater, and Oxia Planum. We doped these samples with a large variety of organic molecules (both biotic and prebiotic molecules) like amino acids, nucleotides, monosaccharides, aldehydes, lipids. We investigated molecular photostability under UV irradiation by monitoring in situ possible modifications of infrared spectroscopic features. These investigations provide pivotal information for ground analysis carried out by rovers on Mars. Introduction Laboratory simulations of Mars are key to support the scientific activity and technology development of life detection instruments on board present and upcoming rover missions such as Mars2020 Perseverance [1] and ExoMars2022 Rosalind Franklin [2]. Studies about the stability of organic molecules in a Martian-like environment allow us to explore the conditions for the preservation of molecular biosignatures and develop models for their degradation in the Martian geological record. A systematic study of the effects of UV radiation on a variety of molecule-mineral complexes mimicking Martian soil can be key for the selection of the most interesting samples to analyse in situ or/and collect for sample return. Testing the sensitivity of different techniques for detection of the diagnostic features of molecular biosignatures embedded into mineral matrices as a function of the molecular concentration helps the choice, design and operation of flight instruments, as well as the interpretation of data collected on the ground during mission operative periods. Methods Experimental analyses were conducted in the Astrobiology Laboratory at INAF-Astrophysical Observatory of Arcetri (Firenze, Italy). Laboratory activities pertain to: (i) synthesis of Mars soil analogues doped with organic compounds that are considered potential molecular biosignatures; (ii) UV-irradiation processing of the Mars soil analogues under Martian-like conditions; and (iii) spectroscopic characterization of the Mars soil analogues. Results Such studies have shown to be very informative in identifying mineral deposits most suitable for preservation of organic compounds, while highlighting the complementarity of different techniques for biomarkers detection, which is critical for ensuring the success of space missions devoted to the search for signs of life on Mars. We will present a series of laboratory results on molecular degradation caused by UV on Mars and possible application to detection of organics by Martian rovers [3,4,5,6]. In detail, we investigated the photostability of several amino acids like glycine, alanine, methionine, valine, tryptophan, phenylalanine, glutamic acid, prebiotic molecules like urea, deoxyribose and glycolaldehyde, and biomarkers like nucleotides and phytane adsorbed on relevant Martian analogs. We monitored the degradation of these molecule-mineral complexes through in situ spectroscopic analysis, investigating the reflectance properties of the samples in the NIR/MIR spectral region. Such spectroscopic characterization of molecular alteration products provides support for two upcoming robotic missions to Mars that will employ NIR spectroscopy to look for molecular biosignatures, through the instruments SuperCam on board Mars 2020, ISEM, Ma_MISS and MicrOmega on board ExoMars 2022. Acknowledgements This research was supported by the Italian Space Agency (ASI) grant agreement ExoMars n. 2017-48-H.0. References [1] Farley K. A. et al. (2020) Space Sci. Rev. 216, 142. [2] Vago, J. L. et al. (2017) Astrobiology 6, 309&#8211;347. [3] Fornaro T. et al. (2013) Icarus 226, 1068&#8211;1085. [4] Fornaro T. et al. (2018) Icarus 313, 38-60. [5] Fornaro T. et al. (2020) Front. Astron. Space Sci. 7:539289. [6] Poggiali G. et al. (2020) Front. Astron. Space Sci. 7:18.

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