, 229:931 | Cite as

High-resolution spatial and temporal analysis of phytoalexin production in oats

  • Yoshihiro Izumi
  • Shin’ichiro KajiyamaEmail author
  • Ryosuke Nakamura
  • Atsushi Ishihara
  • Atsushi Okazawa
  • Eiichiro Fukusaki
  • Yasuo Kanematsu
  • Akio Kobayashi
Original Article


The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor.


Phytoalexin production Oats High-resolution spatial and temporal analysis Laser-assisted single-cell sampling Low-diffuse nanoflow liquid chromatography–electrospray ionization-ion trap mass spectrometry (LC–ESI-MS/MS) Line-scanning fluorescence microscopy 



Liquid chromatography–electrospray ionization-ion trap mass spectrometry


Multiple reaction monitoring


Hypersensitive response


Reactive oxygen species


Programmed cell death


5-(and 6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester


Propidium iodide



The hollow optical fiber used in this experiment was supplied by Dr. Yuji Matsuura (Department of Electrical Communications, Graduate School of Engineering, Tohoku University, Japan). This work was supported by grants from the Promotion of Basic Research Activities for Innovative Biosciences program of BRAIN (Bio-oriented Technology Research Advancement Institution, Japan) and from a Grant-in-Aid for Scientific Research (KAKENHI) in Priority Area “Molecular Nano Dynamics” from the Ministry of Education, Culture, Sports, Science and Technology of Japan to A. Kobayashi.

Supplementary material

425_2008_887_MOESM1_ESM.tif (462 kb)
Fig. S1 Time-dependent imaging by fluorescence microscopy. The fluorescence images of mesophyll cells of an oat leaf treated with distilled water (a-d) and elicitor solution (e-g). Bars = 100 μm. (TIFF 461 kb)
425_2008_887_MOESM2_ESM.tif (1.9 mb)
Fig. S2 Verification of specific emission signals detected using the line-scanning fluorescence microscopy from mesophyll cells. (a) Emission spectra detected from authentic standard solutions of NADPH (red line), NADH (blue line) and NAD+ (brown line). Black line is a result experimentally observed at P 1 (Fig. 6a2). (b) Emission spectra detected for authentic standard solutions of avenanthramides A (red line) and B (blue line). Black line is a result experimentally observed at P 2 (Fig. 6c2). The emission spectra were drawn with appropriate shift in a vertical direction for comparison. (TIFF 1895 kb)


  1. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  2. Böttcher C, Roepenack-Lahaye EV, Willscher E, Scheel D, Clemens S (2007) Evaluation of matrix effects in metabolite profiling based on capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Anal Chem 79:1507–1513PubMedCrossRefGoogle Scholar
  3. Casson S, Spencer M, Walker K, Lindsey K (2005) Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis. Plant J 42:111–123PubMedCrossRefGoogle Scholar
  4. Celis A, Rasmussen HH, Celis P, Basse B, Lauridsen JB, Ratz G, Hein B, Ostergaard M, Wolf H, Orntoft T, Celis JE (1999) Short-term culturing of low-grade superficial bladder transitional cell carcinomas leads to changes in the expression levels of several proteins involved in key cellular activities. Electrophoresis 20:355–361PubMedCrossRefGoogle Scholar
  5. Clarke DD (1982) The accumulation of cinnamic acid amides in the cell walls of potato tissue as an early response to fungal attack. In: Wood RKS (ed) Active defense mechanism in plants. Plenum Press, New York, pp 321–322Google Scholar
  6. Collins FW (1989) Oat phenolics: avenanthramides, novel sbstituted N-cinnamoylanthranilate alkaloids from oat groats and hulls. J Agric Food Chem 37:60–66CrossRefGoogle Scholar
  7. De Block M (1995) In situ enzyme histochemistry on plastic-embedded plant material. Methods Cell Biol 49:153–163PubMedCrossRefGoogle Scholar
  8. Dixon RA (2001) Natural products and plant disease resistance. Nature 411:843–847PubMedCrossRefGoogle Scholar
  9. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274:998–1001PubMedCrossRefGoogle Scholar
  10. Fukusaki E, Harada K, Bamba T, Kobayashi A (2005) An isotope effect on the comparative quantification of flavonoids by means of methylation-based stable isotope dilution coupled with capillary liquid chromatography/mass spectrometry. J Biosci Bioeng 99:75–77PubMedCrossRefGoogle Scholar
  11. Grosset J, Marty I, Chartier Y, Meyer Y (1990) Messenger RNAs newly synthesized by tobacco mesophyll protoplasts are wound inducible. Plant Mol Biol 15:485–496PubMedCrossRefGoogle Scholar
  12. Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H (2001) A large family of class III plant peroxidases. Plant Cell Physiol 42:462–468PubMedCrossRefGoogle Scholar
  13. Inada N, Wildermuth MC (2005) Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta 221:9–16PubMedCrossRefGoogle Scholar
  14. Ishihama Y, Rappsilber J, Andersen JS, Mann M (2002) Microcolumns with self-assembled particle frits for proteomics. J Chromatogr A 979:233–239PubMedCrossRefGoogle Scholar
  15. Ishihara A, Miyagawa H, Matsukawa T, Ueno T, Mayama S, Iwamura H (1998) Induction of hydroxyanthranilate hydroxycinnamoyl transferase activity by oligo-N-acetylchitooligosaccharides in oats. Phytochemistry 47:969–974CrossRefGoogle Scholar
  16. Ishihara A, Ohtsu Y, Iwamura H (1999) Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves. Planta 208:512–518CrossRefGoogle Scholar
  17. Kajiyama S, Harada K, Fukusaki E, Kobayashi A (2006) Single cell-based analysis of torenia petal pigments by a combination of ArF excimer laser micro sampling and nano-high performance liquid chromatography (HPLC)-mass spectrometry. J Biosci Bioeng 102:575–578PubMedCrossRefGoogle Scholar
  18. Kehr J (2001) High resolution spatial analysis of plant systems. Curr Opin Plant Biol 4:197–201PubMedCrossRefGoogle Scholar
  19. Koltai H, Bird DM (2000) High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections. Plant Physiol 123:1203–1212PubMedCrossRefGoogle Scholar
  20. Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275PubMedCrossRefGoogle Scholar
  21. Lange BM (2005) Single-cell genomics. Curr Opin Plant Biol 8:236–241PubMedCrossRefGoogle Scholar
  22. Li SH, Schneider B, Gershenzon J (2007) Microchemical analysis of laser-microdissected stone cells of Norway spruce by cryogenic nuclear magnetic resonance spectroscopy. Planta 225:771–779PubMedCrossRefGoogle Scholar
  23. Mach JM, Castillo AR, Hoogstraten R, Greenberg JT (2001) The Arabidopsis-accelerated cell death gene ACD2 encodes red chlorophyll catabolite reductase and suppresses the spread of disease symptoms. Proc Natl Acad Sci USA 98:771–776PubMedCrossRefGoogle Scholar
  24. Matsuura Y, Miyagi M (1999) Aluminum-coated hollow glass fibers for ArF-excimer laser light fabricated by metallorganic chemical-vapor deposition. Appl Opt 38:2458–2462PubMedCrossRefGoogle Scholar
  25. Medley CD, Drake TJ, Tomasini JM, Rogers RJ, Tan W (2005) Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells. Anal Chem 77:4713–4718PubMedCrossRefGoogle Scholar
  26. Miyagawa H, Ishihara A, Nishimoto T, Ueno T, Mayama S (1995) Induction of avenanthramides in oat leaves inoculated with crown rust fungus, Puccinia coronata f. sp. avenae. Biosci Biotechnol Biochem 59:2305–2306CrossRefGoogle Scholar
  27. Miyagawa H, Ishihara A, Kuwahara Y, Ueno T, Mayama S (1996) Comparative studies of elicitors that induce phytoalexin in oats. J Pesticide Sci 21:203–207Google Scholar
  28. Nakamura R, Izumi Y, Kajiyama S, Kobayashi A, Kanematsu Y (2008) Line-scanning microscopy for time-gated and spectrally-resolved fluorescence imaging. J Biol Phys 34:51–62CrossRefPubMedGoogle Scholar
  29. Ng K-M, Liang Z, Lu W, Tang H-W, Zhao Z, Che C-M, Cheng Y-C (2007) In vivo analysis and spatial profiling of phytochemicals in herbal tissue by matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem 79:2745–2755PubMedCrossRefGoogle Scholar
  30. Okazaki Y, Isobe T, Iwata Y, Matsukawa T, Matsuda F, Miyagawa H, Ishihara A, Nishioka T, Iwamura H (2004) Metabolism of avenanthramide phytoalexins in oats. Plant J 39:560–572PubMedCrossRefGoogle Scholar
  31. Okazaki Y, Ishizuka A, Ishihara A, Nishioka T, Iwamura H (2007) New dimeric compounds of avenanthramide phytoalexin in oats. J Org Chem 72:3830–3839PubMedCrossRefGoogle Scholar
  32. Oosterkamp AJ, Gelpí E, Abian J (1998) Quantitative peptide bioanalysis using column-switching nano liquid chromatography/mass spectrometry. J Mass Spectrom 33:976–983PubMedCrossRefGoogle Scholar
  33. Outlaw WH, Zhang S (2001) Single-cell dissection and microdroplet chemistry. J Exp Bot 52:605–614PubMedCrossRefGoogle Scholar
  34. Peng M, Kuc J (1992) Peroxidase-generated hydrogen peroxide as a source of antifungal activity in vitro and on tobacco leaf disks. Phytopathology 82:696–699CrossRefGoogle Scholar
  35. Pierce ML, Cover EC, Richardson PE, Scholes VE, Essenberg M (1996) Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves. Physiol Mol Plant Pathol 48:305–324CrossRefGoogle Scholar
  36. Ross AR, Ambrose SJ, Cutler AJ, Feurtado JA, Kermode AR, Nelson K, Zhou R, Abrams SR (2004) Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring. Anal Biochem 329:324–333PubMedCrossRefGoogle Scholar
  37. Roy SJ, Cuin TA, Leigh RA (2003) Nanolitre-scale assays to determine the activities of enzymes in individual plant cells. Plant J 34:555–564PubMedCrossRefGoogle Scholar
  38. Schneider B, Hölscher D (2007) Laser microdissection and cryogenic nuclear magnetic resonance spectroscopy: an alliance for cell type-specific metabolite profiling. Planta 225:763–770PubMedCrossRefGoogle Scholar
  39. Sergeeva LI, Vreugdenhil D (2002) In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues. J Exp Bot 53:361–370PubMedCrossRefGoogle Scholar
  40. Shen Y, Tolić N, Masselon C, Paša-Tolić L, Camp DG 2nd, Hixson KK, Zhao R, Anderson GA, Smith RD (2004) Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS. Anal Chem 76:144–154PubMedCrossRefGoogle Scholar
  41. Torres MA, Dangl JL, Jones JDG (2002) Arabidopsis gp91 phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522PubMedCrossRefGoogle Scholar
  42. Wada M, Kagawa T, Sato Y (2003) Chloroplast movement. Annu Rev Plant Biol 54:455–468PubMedCrossRefGoogle Scholar
  43. Wahl JH, Goodlett DR, Udseth HR, Smith RD (1993) Use of small-diameter capillaries for increasing peptide and protein detection sensitivity in capillary electrophoresis-mass spectrometry. Electrophoresis 14:448–457PubMedCrossRefGoogle Scholar
  44. Yao N, Greenberg JT (2006) Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death. Plant Cell 18:397–411PubMedCrossRefGoogle Scholar
  45. Zhao J, Fujita K, Sakai K (2007) Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis. New Phytol 175:215–229PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Yoshihiro Izumi
    • 2
  • Shin’ichiro Kajiyama
    • 1
    • 2
    Email author
  • Ryosuke Nakamura
    • 4
    • 5
  • Atsushi Ishihara
    • 3
  • Atsushi Okazawa
    • 2
  • Eiichiro Fukusaki
    • 2
  • Yasuo Kanematsu
    • 4
  • Akio Kobayashi
    • 2
  1. 1.School of Biology-Oriented Science and TechnologyKinki UniversityKinokawaJapan
  2. 2.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan
  3. 3.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversityKyotoJapan
  4. 4.JST-CREST, Venture Business Laboratory, Center for Advanced Science and InnovationOsaka UniversitySuitaJapan
  5. 5.Department of Physics, Graduate School of ScienceTohoku UniversitySendaiJapan

Personalised recommendations