Rapid determination of spore chemistry using thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy

Abstract

Spore chemistry is at the centre of investigations aimed at producing a proxy record of harmful ultraviolet radiation (UV-B) through time. A biochemical proxy is essential owing to an absence of long-term (century or more) instrumental records. Spore cell material contains UV-B absorbing compounds that appear to be synthesised in variable amounts dependent on the ambient UV-B flux. To facilitate these investigations we have developed a rapid method for detecting variations in spore chemistry using combined thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy. Our method was tested using spores obtained from five populations of the tropical lycopsid Lycopodium cernuum growing across an altitudinal gradient (650-1981 m a.s.l.) in S.E. Asia with the assumption that they experienced a range of UV-B radiation doses. Thermochemolysis and subsequent pyrolysis liberated UV-B pigments (ferulic and para-coumaric acid) from the spores. All of the aromatic compounds liberated from spores by thermochemolysis and pyrolysis were active in UV-B protection. The various functional groups associated with UV-B protecting pigments were rapidly detected by micro-FTIR and included the aromatic C[double bond, length as m-dash]C absorption band which was exclusive to the pigments. We show increases in micro-FTIR aromatic absorption (1510 cm(-1)) with altitude that may reflect a chemical response to higher UV-B flux. Our results indicate that rapid chemical analyses of historical spore samples could provide a record ideally suited to investigations of a proxy for stratospheric O3 layer variability and UV-B flux over historical (century to millennia) timescales.

The impact of oxidation on spore and pollen chemistry

“…Samples treated with cold acetolysis show only subtle differences to untreated material (Figs 2A and S1). There are slight positive and negative shifts in absorbance peaks between 900 and 1300 cm -1 that probably represent C-O bonds (Watson et al, 2007), and a slight decrease in the aliphatic peaks at 2925 and 2850 cm -1 , but there are no clear trends with increasing treatment durations. Applying heat to the acetolysis treatment (Figs 2B and S1) produces an immediate chemical shift with several peaks decreasing in size, most notably the aliphatic peaks but also the 1710 cm -1 carboxyl peak, the 1510 cm -1 aromatic b peak, and C-O peaks at 1100 and 980 cm -1 .…”

“…Applying heat (Figs 2D and S1) speeds up the chemical changes considerably, with a large increase in both the carboxyl and aromatic b peaks; the aromatic b peak also shifts position to c. 1550 cm -1 . The aromatic a peak disappears completely, and there is a large decrease in the size of the C-O peaks at c. 1120 and 990 cm -1 , and an ester peak at 1740 cm -1 (Watson et al, 2007). Measurement interval is the interval within which each band height was measured, taken as the maximum value within that interval.…”

“…Measurement interval is the interval within which each band height was measured, taken as the maximum value within that interval. Band assignments follow Watson et al (2007) and Fraser et al (2011Fraser et al ( , 2014b. v = stretching; as = asymmetrical.…”

“…We use ratios between peak heights to characterize sporopollenin chemistry, again to remove the potential impact of differing sample thickness on absolute absorbance values. UV-B absorbing compounds are detected in IR spectra via an absorbance peak that occurs near 1510 cm -1 , caused by the stretching by guest on May 10, 2018 http://jm.lyellcollection.org/ Downloaded from of C=C bonds in the aromatic ring structure (Watson et al, 2007;Fraser et al, 2014b), and have previously been quantified via a ratio with the broad hydroxyl absorbance band that centres on 3300 cm -1 (Fig. 1) (Lomax et al, 2008Fraser et al, 2011).…”

Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Pollen Development

“…Sporopollenin is thought to be composed entirely of polyalkyl and aromatic macromolecules as well as aliphatic and aromatic monomers, particularly ferulic and p-coumaric acids that are hinged together (Guilford et al, 1988;Wehling et al, 1989;Mösle et al, 1997;Blokker et al, 2005;De Leeuw et al, 2006;Watson et al, 2007). Intine, the innermost layer of the pollen wall, mainly consists of pectin, cellulose, hemicellulose (Kress and Stone, 1983;Edlund et al, 2004;Fang et al, 2008), and also of hydroxycinnamic acids (Meychik et al, 2006).…”