The Plant Journal (2009) 60, 907–918
doi: 10.1111/j.1365-313X.2009.04012.x
TECHNICAL ADVANCE
Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species Dirk Ho¨lscher1,*, Rohit Shroff1, Katrin Knop2, Michael Gottschaldt2,3, Anna Crecelius2,3, Bernd Schneider1, David G. Heckel1, Ulrich S. Schubert2,3,4 and Alesˇ Svatosˇ1,* 1 Department of Entomology, Mass Spectrometry Research Group, and Biosynthesis/NMR Research Group, Max Planck Institute for Chemical Ecology, Hans-Kno¨ll-Strasse 8, 07745 Jena, Germany, 2 Laboratory of Organic and Macromolecular Chemistry, Friedrich-Schiller University of Jena, Humboldtstrasse 10, 07743 Jena, Germany, 3 Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB Eindhoven, The Netherlands, and 4 Laboratory of Macromolecular Chemistry and Nanoscience, Eindhoven University of Technology, Den Dolech 2, 5600 MB Eindhoven, The Netherlands Received 8 May 2009; revised 8 August 2009; accepted 24 August 2009; published online 27 October 2009. * For correspondence (fax +49 3641 571701; e-mail
[email protected] or
[email protected]).
SUMMARY The present paper describes matrix-free laser desorption/ionisation mass spectrometric imaging (LDI-MSI) of highly localized UV-absorbing secondary metabolites in plant tissues at single-cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 lm, was much higher than that achieved by commonly used matrix-assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI-TOF/MS analysis of cryo-sectioned, laser-microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI-TOF/MS or LDI-MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI-MSI for other commonly occurring compounds such as flavonoids. The organspecific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level. Keywords: Hypericum perforatum, Hypericum reflexum, LDI mass spectrometric imaging, laser microdissection, naphthodianthrones, flavonoids.
INTRODUCTION Recently, a variety of technologies have been successfully used to obtain data from individual plant cells (Lange, 2005). Both laser microdissection (LMD) (Emmert-Buck et al., 1996; Ho¨lscher and Schneider, 2007; Li et al., 2007a) and cell sap sampling using microcapillaries (Brandt et al., 2002) have been reported to improve access to the contents of individual cells; these reports were based on analyses using both post-genomic bioanalytical technologies and spectroscopic ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd
methods (Lange, 2005). Techniques such as LMD allow researchers to avoid the averaging effects that occur when heterogeneous tissues, which represent the most abundant cell types, are pooled; however, as amplification methods such as commonly used for DNA and RNA are not available, highly sensitive detection methods are required for metabolites. Methods based on mass spectrometry offer the required high sensitivity and specificity (Sumner et al., 2003). 907
908 Dirk Ho¨lscher et al. MS-dependent imaging techniques have been developed in the past to study the localization on a small scale of compounds from complex biological systems. Recently, a method for MS imaging has been developed that offers information about the distribution of proteins, peptides or metabolites with 100–300 lm spatial resolution (Caprioli et al., 1997; Cooks et al., 2006; Li et al., 2008; Seeley and Caprioli, 2008). Furthermore, when atmospheric pressure infrared matrix-assisted laser desorption/ionization mass spectrometry was used to study plant metabolites in the white lily (Lilium candidum L.) and other plants, over 50 small metabolites were discovered; these were involved in flavonoid biosynthesis and had a spatial resolution of 180– 640 lm (Li et al., 2008). MALDI imaging involves use of a conventional MALDI source to desorb ions of interest from the sample covered by a sprayed (Rubakhin et al., 2005) or spotted matrix (Aerni et al., 2006). MALDI imaging has been used to study the distribution of a wide variety of metabolites, including drugs, peptides and proteins, in animal tissues (Reyzer and Caprioli, 2007; Goodwin et al., 2008), and herbicides (Mullen et al., 2005), peptides (Kondo et al., 2006) and sugars (Burrell et al., 2007; Li et al., 2007a,b) in plants. Recently, ion intensity maps were constructed from MALDITOF mass spectra to measure the spatial distribution of some secondary plant metabolites. The matrix 9-aminoacridine was evenly applied to the leaves of Arabidopsis thaliana (Col-0) in order to detect glucosinolates (Shroff et al., 2008). However, applying MALDI matrices to the tissues complicates tissue preparation for imaging and can disturb the native distribution of the studied metabolites
(Shroff et al., 2008). Low-molecular-weight metabolites have been profiled and localized using colloidal graphite-assisted LDI (GALDI) MS in A. thaliana (Cha et al., 2008). Profiles and spatially resolved images of phospholipids, cerebrosides, oligosaccharides, flavonoids and other secondary metabolites were obtained in negative and positive ion MS modes. Certain mass imaging methods, such as laser-assisted electrospray ionization (LAESI) (Vertes et al., 2008), can reduce sample handling prior to analysis. However, the infrared laser currently used does not provide cell-like resolution for the obtained MS images. The main secondary plant products of members of the genus Hypericum of the Hypericaceae plant family (Mabberley, 2008) are flavonoids (e.g. quercetin, hyperoside, rutin, quercitrin, isoquercitrin), xanthones (e.g. 1,3,6,7,tetrahydroxyxanthone), prenylated phloroglucinols such as hyperforin and adhyperforin, biflavonoids [I3,II8-biapigenin and I3¢,II8-biapigenin (amentoflavone)] and naphthodianthrones (Brockmann et al., 1942; Wolfender et al., 2003; Charchogylan et al., 2007; Smelcerovic et al., 2008) (Figure 1 and Table 1). Multi-cellular, globular or tunnel-shaped aggregates such as secretory canals, and dark and translucent glands have been reported to contain the secondary metabolites (Ciccarelli et al., 2001; Robson, 2003). Dark and pale glands often occur in the stem, leaf, sepal, petal or anthers. In Hypericum, the stamens possess a connective terminating in a dark gland (Figure 2). The gynoecium of H. perforatum has three styles and contains relatively small red stigmata (Figure 2). There is a large variation in the glandularity of sepals in Hypericum, and some species (such
Figure 1. Chemical structures for the major secondary metabolites of H. perforatum.
ª 2009 The Authors Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 907–918
Matrix-free UV-LDI mass spectrometric imaging at the single-cell level 909 Table 1 Names, deprotonated ion mass and UV adsorption wavelengths of the Hypericum sp. natural constituents (see Figure 1 for structural formula) (Ju¨rgenliemk, 2001)
Number
Name
[M-H]) (m/z)
UVmax (nm)
(1) (2) (3) (4) (5) (6) (7)
Quercetin Quercitrin (R: 3-O-a-L-rhamnopyranosyl) Isoquercitrin (R: 3-O-b-D-glucopyranosyl) Hyperoside (R: 3-O-b-D-galactopyranosyl) Miquelianin (R: 3-O-a-D-glucuronopyranosyl) Rutin (R: 3-O-b-D-rutinosyl) Hypericin (R: -CH3)
301.03 447.09 463.08 463.08 477.07 609.14 503.07
(8)
Pseudohypericin (R: -CH2OH)
519.07
(9) (10) (11) (12) (13) (14) (15)
Protohypericin (R: -CH3) Protopseudohypericin (R: -CH2OH) Hyperforin (R: -H) Adhyperfirin (R: -CH3) Hyperfirin (R: -H) Biapigenin (I3,II8-biapigenin) Amentoflavone (I3¢,II8-biapigenin)
505.09 521.08 535.37 481.33 467.31 537.08 537.08
256.8, 372.9 256.8, 349.3 256.8, 352.9 256.8, 352.9 256.8, 352.9 256.8, 352.9 278.1, 317.2, 452.3, 539.5, 579.7 281.6, 324.3, 455.9, 539.5, 579.7 – – 271.0 – – 267.4, 331.4 –
as H. reflexum) have stalked glands (Robson, 2003). These glandular trichomes are often close neighbours of dark glands, which are located near the margins of the sepals (Esau, 1965; Piovan et al., 2004). The short distance (
