Abstract
Main conclusion
FTIR microspectroscopy, in combination with spectral averaging procedure, enables precise analysis of pollen grains for chemical characterization and identification studies of fresh and fossilised pollen in botany, ecology and palaeosciences.
Infrared microspectroscopy (µFTIR) of Pinaceae pollen can provide valuable information on plant phenology, ecophysiology and paleoecology, but measurements are challenging, resulting in unreproducible spectra. The comparative analysis of µFTIR spectra belonging to morphologically different Pinaceae pollen, namely bisaccate Pinus and monosaccate Tsuga pollen, was conducted. The study shows that the main cause of spectral variability is non-radial symmetry of bisaccate pollen grains, while additional variation is caused by Mie scattering. Averaging over relatively small number of single pollen grain spectra (approx. 5–10) results with reproducible data on pollen chemical composition. The practical applicability of the µFTIR spectral averaging method has been demonstrated by the partial least-squares regression-based differentiation of the two closely related Pinus species with morphologically indistinguishable pollen: Pinus mugo (mountain pine) and Pinus sylvestris (Scots pine). The study has demonstrated that the µFTIR approach can be used for identification, differentiation and chemical characterization of pollen with complex morphology. The methodology enables analysis of fresh pollen, as well as fossil pollen from sediment core samples, and can be used in botany, ecology and paleoecology for study of biotic and abiotic effects on plants.
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References
Ariizumi T, Toriyama K (2011) Genetic regulation of sporopollenin synthesis and pollen exine development. Annu Rev Plant Biol 62:437–460. doi:10.1146/annurev-arplant-042809-112312
Bağcıoğlu M, Zimmermann B, Kohler A (2015) A multiscale vibrational spectroscopic approach for identification and biochemical characterization of pollen. Plos One 10(9):e0137899. doi:ARTN10.1371/journal.pone.0137899
Bağcıoğlu M, Kohler A, Seifert S, Kneipp J, Zimmermann B (2017) Monitoring of plant—environment interactions by high-throughput FTIR spectroscopy of pollen. Methods Ecol Evol 8(7):870–880. doi:10.1111/2041-210X.12697
Bedinger P (1992) The remarkable biology of pollen. Plant Cell 4(8):879–887. doi:10.1105/tpc.4.8.879
Blackmore S, Wortley AH, Skvarla JJ, Rowley JR (2007) Pollen wall development in flowering plants. New Phytol 174(3):483–498. doi:10.1111/j.1469-8137.2007.02060.x
Blokker P, Yeloff D, Boelen P, Broekman RA, Rozema J (2005) Development of a proxy for past surface UV-B irradiation: a thermally assisted hydrolysis and methylation py-GC/MS method for the analysis of pollen and spores. Anal Chem 77(18):6026–6031. doi:10.1021/ac050696k
Boratynska K, Jasinska AK, Boratynski A (2015) Taxonomic and geographic differentiation of Pinus mugo complex on the needle characteristics. Syst Biodivers 13(6):901–915. doi:10.1080/14772000.2015.1058300
Boyain-Goitia AR, Beddows DCS, Griffiths BC, Telle HH (2003) Single-pollen analysis by laser-induced breakdown spectroscopy and Raman microscopy. Appl Opt 42(30):6119–6132. doi:10.1364/Ao.42.006119
Businsky R, Kirschner J (2006) Nomenclatural notes on the Pinus mugo complex in Central Europe. Phyton Ann Rei Bot A 46(1):129–139
Bykowska J, Klimko M (2015) Pollen morphology of Pinus mugo Turra × Pinus sylvestris L. hybrids and parental species in an experimental culture. Acta Biol Cracoviensia Bot 57(1):149–160. doi:10.1515/abcsb-2015-0009
Christensen KI (1987) Taxonomic revision of the Pinus mugo complex and P. rhaetica (P. rhaetica-mugo × sylvestris) (Pinaceae). Nord J Bot 7(4):383–408. doi:10.1111/j.1756-1051.1987.tb00958.x
Christensen KI, Dar GH (1997) A morphometric analysis of spontaneous and artificial hybrids of Pinus mugo × sylvestris (Pinaceae). Nord J Bot 17(1):77–86. doi:10.1111/j.1756-1051.1997.tb00291.x
Dell’Anna R, Lazzeri P, Frisanco M, Monti F, Malvezzi Campeggi F, Gottardini E, Bersani M (2009) Pollen discrimination and classification by Fourier transform infrared (FT-IR) microspectroscopy and machine learning. Anal Bioanal Chem 394(5):1443–1452. doi:10.1007/s00216-009-2794-9
Depciuch J, Kasprzyk I, Roga E, Parlinska-Wojtan M (2016) Analysis of morphological and molecular composition changes in allergenic Artemisia vulgaris L. pollen under traffic pollution using SEM and FTIR spectroscopy. Environ Sci Pollut R 23(22):23203–23214. doi:10.1007/s11356-016-7554-8
Depciuch J, Kasprzyk I, Sadik O, Parlinska-Wojtan M (2017) FTIR analysis of molecular composition changes in hazel pollen from unpolluted and urbanized areas. Aerobiologia 33(1):1–12. doi:10.1007/s10453-016-9445-3
Dominguez E, Mercado JA, Quesada MA, Heredia A (1999) Pollen sporopollenin: degradation and structural elucidation. Sex Plant Reprod 12(3):171–178. doi:10.1007/s004970050189
Fraser WT, Sephton MA, Watson JS, Self S, Lomax BH, James DI, Wellman CH, Callaghan TV, Beerling DJ (2011) UV-B absorbing pigments in spores: biochemical responses to shade in a high-latitude birch forest and implications for sporopollenin-based proxies of past environmental change. Polar Res 30:8312. doi:10.3402/polar.v30i0.8312
Fraser WT, Scott AC, Forbes AES, Glasspool IJ, Plotnick RE, Kenig F, Lomax BH (2012) Evolutionary stasis of sporopollenin biochemistry revealed by unaltered Pennsylvanian spores. New Phytol 196(2):397–401. doi:10.1111/j.1469-8137.2012.04301.x
Fraser WT, Watson JS, Sephton MA, Lomax BH, Harrington G, Gosling WD, Self S (2014) Changes in spore chemistry and appearance with increasing maturity. Rev Palaeobot Palynol 201:41–46. doi:10.1016/j.revpalbo.2013.11.001
Gottardini E, Rossi S, Cristofolini F, Benedetti L (2007) Use of Fourier transform infrared (FT-IR) spectroscopy as a tool for pollen identification. Aerobiologia 23(3):211–219
Hesse M (2009) Pollen terminology: an illustrated handbook. Springer, Wien
Ho R, Sziklai O (1972) On the pollen morphology of Picea and Tsuga species. Grana 12(1):31–40. doi:10.1080/00173137209427643
Holmes-Davis R, Tanaka CK, Vensel WH, Hurkman WJ, McCormick S (2005) Proteome mapping of mature pollen of Arabidopsis thaliana. Proteomics 5(18):4864–4884. doi:10.1002/pmic.200402011
Holt KA, Bennett KD (2014) Principles and methods for automated palynology. New Phytol 203(3):735–742. doi:10.1111/nph.12848
Jardine PE, Fraser WT, Lomax BH, Sephton MA, Shanahan TM, Miller CS, Gosling WD (2016) Pollen and spores as biological recorders of past ultraviolet irradiance. Sci Rep 6:39269. doi:10.1038/srep39269
Jardine PE, Abernethy FAJ, Lomax BH, Gosling WD, Fraser WT (2017) Shedding light on sporopollenin chemistry, with reference to UV reconstructions. Rev Palaeobot Palynol 238:1–6. doi:10.1016/j.revpalbo.2016.11.014
Jiang J, Zhang Z, Cao J (2013) Pollen wall development: the associated enzymes and metabolic pathways. Plant Biol 15(2):249–263. doi:10.1111/j.1438-8677.2012.00706.x
Jiang YF, Lahlali R, Karunakaran C, Kumar S, Davis AR, Bueckert RA (2015) Seed set, pollen morphology and pollen surface composition response to heat stress in field pea. Plant Cell Environ 38(11):2387–2397. doi:10.1111/pce.12589
Joester M, Seifert S, Emmerling F, Kneipp J (2016) Physiological influence of silica on germinating pollen as shown by Raman spectroscopy. J Biophotonics. doi:10.1002/jbio.201600011
Julier ACM, Jardine PE, Coe AL, Gosling WD, Lomax BH, Fraser WT (2016) Chemotaxonomy as a tool for interpreting the cryptic diversity of Poaceae pollen. Rev Palaeobot Palynol 235:140–147. doi:10.1016/j.revpalbo.2016.08.004
Kim SS, Douglas CJ (2013) Sporopollenin monomer biosynthesis in Arabidopsis. J Plant Biol 56(1):1–6. doi:10.1007/s12374-012-0385-3
Klaus W (1978) On the taxonomic significance of tectum sculpture characters in alpine Pinus species. Grana 17(3):161–166. doi:10.1080/00173137809431961
Lahlali R, Jiang YF, Kumar S, Karunakaran C, Liu X, Borondics F, Hallin E, Bueckert R (2014) ATR-FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance. Front Plant Sci 5:747. doi:10.3389/fpls.2014.00747
Liang M, Zhang P, Shu X, Liu CG, Shu JN (2013) Characterization of pollen by MALDI-TOF lipid profiling. Int J Mass Spectrom 334:13–18. doi:10.1016/j.ijms.2012.09.007
Lindbladh MS, O’Connor R, Jacobson GL (2002) Morphometric analysis of pollen grains for paleoecological studies: classification of Picea from eastern North America. Am J Bot 89(9):1459–1467. doi:10.3732/ajb.89.9.1459
Lomax BH, Fraser WT (2015) Palaeoproxies: botanical monitors and recorders of atmospheric change. Palaeontology 58(5):759–768. doi:10.1111/pala.12180
Lomax BH, Fraser WT, Sephton MA, Callaghan TV, Self S, Harfoot M, Pyle JA, Wellman CH, Beerling DJ (2008) Plant spore walls as a record of long-term changes in ultraviolet-B radiation. Nat Geosci 1(9):592–596. doi:10.1038/ngeo278
Lomax BH, Fraser WT, Harrington G, Blackmore S, Sephton MA, Harris NBW (2012) A novel palaeoaltimetry proxy based on spore and pollen wall chemistry. Earth Planet Sci Lett 353:22–28. doi:10.1016/j.epsl.2012.07.039
Lukacs R, Blumel R, Zimmermann B, Bagcoglu M, Kohler A (2015) Recovery of absorbance spectra of micrometer-sized biological and inanimate particles. Analyst 140:3273–3278. doi:10.1039/C5AN00401B
Miller CN (1999) Implications of fossil conifers for the phylogenetic relationships of living families. Bot Rev 65(3):239–277. doi:10.1007/Bf02857631
Nakagawa T, Edouard JL, de Beaulieu JL (2000) A scanning electron microscopy (SEM) study of sediments from Lake Cristol, southern French Alps, with special reference to the identification of Pinus cembra and other Alpine Pinus species based on SEM pollen morphology. Rev Palaeobot Palynol 108(1–2):1–15. doi:10.1016/S0034-6667(99)00030-5
Nakazawa F, Uetake J, Suyama Y, Kaneko R, Takeuchi N, Fujita K, Motoyama H, Imura S, Kanda H (2013) DNA analysis for section identification of individual Pinus pollen grains from Belukha glacier, Altai Mountains, Russia. Environ Res Lett 8:014032. doi:10.1088/1748-9326/8/1/014032
Pacini E (1996) Types and meaning of pollen carbohydrate reserves. Sex Plant Reprod 9(6):362–366. doi:10.1007/Bf02441957
Pacini E, Hesse M (2005) Pollenkitt—its composition, forms and functions. Flora 200(5):399–415. doi:10.1016/j.flora.2005.02.006
Pappas CS, Tarantilis PA, Harizanis PC, Polissiou MG (2003) New method for pollen identification by FT-IR spectroscopy. Appl Spectrosc 57(1):23–27
Parducci L, Suyama Y, Lascoux M, Bennett KD (2005) Ancient DNA from pollen: a genetic record of population history in Scots pine. Mol Ecol 14(9):2873–2882. doi:10.1111/j.1365-294X.2005.02644.x
Parodi G, Dickerson P, Cloud J (2013) Pollen identification by Fourier transform infrared photoacoustic spectroscopy. Appl Spectrosc 67(3):342–348. doi:10.1366/12-06622
Piffanelli P, Ross JHE, Murphy DJ (1998) Biogenesis and function of the lipidic structures of pollen grains. Sex Plant Reprod 11(2):65–80. doi:10.1007/s004970050122
Pummer BG, Bauer H, Bernardi J, Chazallon B, Facq S, Lendl B, Whitmore K, Grothe H (2013) Chemistry and morphology of dried-up pollen suspension residues. J Raman Spectrosc 44(12):1654–1658. doi:10.1002/jrs.4395
Roulston TH, Cane JH, Buchmann SL (2000) What governs protein content of pollen: pollinator preferences, pollen-pistil interactions, or phylogeny? Ecol Monogr 70(4):617–643. doi:10.1890/0012-9615(2000)070[0617:Wgpcop]2.0.Co;2
Rozema J, Broekman RA, Blokker P, Meijkamp BB, de Bakker N, van de Staaij J, van Beem A, Ariese F, Kars SM (2001) UV-B absorbance and UV-B absorbing compounds (para-coumaric acid) in pollen and sporopollenin: the perspective to track historic UV-B levels. J Photochem Photobiol B 62(1–2):108–117. doi:10.1016/S1011-1344(01)00155-5
Savolainen O, Pyhajarvi T, Knurr T (2007) Gene flow and local adaptation in trees. Annu Rev Ecol Evol S 38:595–619. doi:10.1146/annurev.ecolsys.38.091206.095646
Seifert S, Weidner SM, Panne U, Kneipp J (2015) Taxonomic relationships of pollens from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data using multivariate statistics. Rapid Commun Mass Spectrom 29(12):1145–1154. doi:10.1002/rcm.7207
Seifert S, Merk V, Kneipp J (2016) Identification of aqueous pollen extracts using surface enhanced Raman scattering (SERS) and pattern recognition methods. J Biophotonics 9(1–2):181–189. doi:10.1002/jbio.201500176
Shi JX, Cui MH, Yang L, Kim YJ, Zhang DB (2015) Genetic and biochemical mechanisms of pollen wall development. Trends Plant Sci 20(11):741–753. doi:10.1016/j.tplants.2015.07.010
Souza CD, Kim SS, Koch S, Kienow L, Schneider K, McKim SM, Haughn G, Kombrink E, Douglas CJ (2009) A novel fatty acyl-CoA synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis. Plant Cell 21(2):507–525. doi:10.1105/tpc.108.062513
Sowa S, Connor KF, Towill LE (1991) Temperature changes in lipid and protein structure measured by Fourier transform infrared spectrophotometry in intact pollen grains. Plant Sci 78(1):1–9. doi:10.1016/0168-9452(91)90155-2
Speranza A, Calzoni GL, Pacini E (1997) Occurrence of mono- or disaccharides and polysaccharide reserves in mature pollen grains. Sex Plant Reprod 10(2):110–115. doi:10.1007/s004970050076
Staszkie J, Tyszkiew M (1969) Natural hybrids of Pinus mugo Turra and Pinus sylvestris L in Nowy Targ basin. B Acad Pol Sci Biol 17(9):579
Vanherpen MMA (1981) Effect of season, age and temperature on the protein pattern of pollen and styles in Petunia hybrida. Acta Bot Neerl 30(4):277–287
Vesprini JL, Nepi M, Cresti L, Guarnieri M, Pacini E (2002) Changes in cytoplasmic carbohydrate content during Helleborus pollen presentation. Grana 41(1):16–20. doi:10.1080/00173130260045459
Wachowiak W, Palme AE, Savolainen O (2011) Speciation history of three closely related pines Pinus mugo (T.), P. uliginosa (N.) and P. sylvestris (L.). Mol Ecol 20(8):1729–1743. doi:10.1111/j.1365-294X.2011.05037.x
Wachowiak W, Trivedi U, Perry A, Cavers S (2015) Comparative transcriptomics of a complex of four European pine species. BMC Genom 16:234. doi:10.1186/s12864-015-1401-z
Watson JS, Sephton MA, Sephton SV, Self S, Fraser WT, Lomax BH, Gilmour I, Wellman CH, Beerling DJ (2007) Rapid determination of spore chemistry using thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy. Photochem Photobiol Sci 6(6):689–694. doi:10.1039/b617794h
Willis KJ, Feurdean A, Birks HJ, Bjune AE, Breman E, Broekman R, Grytnes JA, New M, Singarayer JS, Rozema J (2011) Quantification of UV-B flux through time using UV-B-absorbing compounds contained in fossil Pinus sporopollenin. New Phytol 192(2):553–560. doi:10.1111/j.1469-8137.2011.03815.x
Wolkers WF, Hoekstra FA (1995) Aging of dry desiccation-tolerant pollen does not affect protein secondary structure. Plant Physiol 109(3):907–915
Yazdani R, Lindgren D, Stewart S (1989) Gene dispersion within a population of Pinus sylvestris. Scand J For Res 4(1–4):295–306. doi:10.1080/02827588909382567
Yule BL, Roberts S, Marshall JEA (2000) The thermal evolution of sporopollenin. Org Geochem 31(9):859–870. doi:10.1016/S0146-6380(00)00058-9
Zhang DB, Yang XJ, Shi JX (2016) Role of lipids in plant pollen development. In: Nakamura Y (ed) Lipids in plant and algae development. Springer, Berlin, pp 315–337
Zimmermann B (2010) Characterization of pollen by vibrational spectroscopy. Appl Spectrosc 64(12):1364–1373
Zimmermann B, Kohler A (2013) Optimizing Savitzky–Golay parameters for improving spectral resolution and quantification in infrared spectroscopy. Appl Spectrosc 67(8):892–902
Zimmermann B, Kohler A (2014) Infrared spectroscopy of pollen identifies plant species and genus as well as environmental conditions. PLoS One 9(4):e95417. doi:10.1371/journal.pone.0095417
Zimmermann B, Bagcioglu M, Sandt C, Kohler A (2015a) Vibrational microspectroscopy enables chemical characterization of single pollen grains as well as comparative analysis of plant species based on pollen ultrastructure. Planta 242(5):1237–1250. doi:10.1007/s00425-015-2380-7
Zimmermann B, Tkalcec Z, Mesic A, Kohler A (2015b) Characterizing aeroallergens by infrared spectroscopy of fungal spores and pollen. PLoS One 10(4):e0124240. doi:10.1371/journal.pone.0124240
Zimmermann B, Tafintseva V, Bagcioglu M, Hoegh Berdahl M, Kohler A (2016) Analysis of allergenic pollen by FTIR microspectroscopy. Anal Chem 88(1):803–811. doi:10.1021/acs.analchem.5b03208
Acknowledgements
The research was supported by the European Commission through the Seventh Framework Programme (FP7-PEOPLE-2012-IEF Project no. 328289). The author thanks M. Furlan Zimmermann.
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Zimmermann, B. Chemical characterization and identification of Pinaceae pollen by infrared microspectroscopy. Planta 247, 171–180 (2018). https://doi.org/10.1007/s00425-017-2774-9
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DOI: https://doi.org/10.1007/s00425-017-2774-9