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Spatial profiling of maytansine during the germination process of Maytenus senegalensis seeds
Fitoterapia 119 (2017) 51–56
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Spatial profiling of maytansine during the germination process of Maytenus senegalensis seeds
MARK
Dennis Eckelmann, Souvik Kusari⁎, Michael Spiteller⁎ Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Maytansine MALDI-imaging-HRMS Seeds Seedlings Chemical defense Maytenus senegalensis
The ecological role of maytansine, an important antineoplastic and antimicrobial compound with high cytotoxicity, particularly as a chemical defense compound has remained elusive since its discovery in the 1970s in Maytenus and Putterlickia plants. In the present study, we have used MALDI-imaging-HRMS to visualize the occurrence as well as spatial and temporal distribution of maytansine in a Maytenus senegalensis plant, seeds obtained from the mother plant during seeding stage, through the germination of the seeds, and finally up to the establishment of seedlings (or daughter plants). Although the mother plant was devoid of maytansine, the bioactive compound was found to be distributed in the cotyledons and the endosperm of the seeds with an augmented accretion towards the seed coat. Furthermore, maytansine was always detected in the emerging seedlings, particularly the cortex encompassing the radicle, hypocotyl, and epicotyl. The typical pattern of accumulation of maytansine not only in the seeds but also during germination provides a proof-of-concept that M. senegalensis is ecologically primed to trigger the production of maytansine in vulnerable tissues such as seeds during plant reproduction. By utilizing maytansine as chemical defense compound against predators and/or pathogens, the plant can ensure viability of the seeds and successful germination, thus leading to the next generation of daughter plants.
1. Introduction In every ecological niche, plants coexist with a plethora of macroand microorganisms. The so-called ‘mutualistic links’ connect plants with associated microorganisms (such as endophytes and invading pathogens) and predators (such as insects and herbivores) with defined costs and fitness [1]. Concomitantly, plants coevolve physical, chemical and molecular means of defense against predators and pathogens, which are crucial not only for their existence but also for delivering vital eco-specific functions [2–5]. Notably, plants are ecologically primed to protect their seeds in order to ensure their continued existence through their offspring. This is emphasized by the fact that seed viability and germination is typically influenced not only by abiotic environmental factors but also by biotic stressors such as predators and pathogens [6–10]. In addition to structural or physical barriers, seeds often contain a repertoire of chemical defense compounds to impede invading predators and/or pathogens. For example, seeds of Phaseolus vulgaris have been shown to contain vulgarinin, an antifungal peptide highly active against fungal phytopathogens Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and
⁎
Botrytis cinerea [11]. In another study, Phaseolus mungo (syn. Vigna mungo) seeds have been reported to contain an antifungal and antibacterial lysozyme [12]. Maytansine (Fig. 1) is an important anticancer, antimicrobial, and cytotoxic compound [13–16], which was first isolated by Kupchan and coworkers from Maytenus and Putterlickia plants (Celastraceae) in the 1970s [17–18]. Owing to its remarkably bioactivity, maytansine garnered immense attention of scientists worldwide, particularly in the prospection of different Celastraceae plants from diverse ecological niches [19]. These intensive efforts led to the isolation of maytansine along with its derivatives sporadically from different plants [20–23]. Additionally, some maytansine analogs were also found in other unrelated organisms such as mosses [24–25] as well as the bacteria Actinosynnema pretiosum [26]. Recently, it was established by our group that in Putterlickia verrucosa and Putterlickia retrospinosa plants, maytansine is actually biosynthesized by eco-specific root-associated endophytic bacterial community [27]. More recently, we demonstrated that the biosynthesis of maytansine in Maytenus serrata prospected from Cameroon is shared between the endophytic bacterial community colonizing the stem and the host plant containing non-culturable
Corresponding authors. E-mail addresses: [email protected] (S. Kusari), [email protected] (M. Spiteller).
http://dx.doi.org/10.1016/j.fitote.2017.03.014 Received 24 February 2017; Received in revised form 27 March 2017; Accepted 30 March 2017 Available online 03 April 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
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this plant along with its seeds by MALDI-imaging-HRMS. In addition, in order to explore whether the spatial distribution of maytansine in M. senegalensis seeds might be representative in other Maytenus species, we bioprospected Maytenus heterophylla seeds from a mature plant in Grahamstown (S33°14,026′; E26°24,817′) in South Africa in February 2017. The prospecting of M. heterophylla seeds was realized with the help of Mr. Ulrich Feiter, Parceval Pty. Ltd., Wellington, South Africa. We analyzed the crude extract of the seeds by HPLC-HRMS and seed sections by MALDI-imaging-HRMS. 2.2. Plant extraction and HPLC-HRMS The extraction of different plant tissues (root, twig, leaf) of the M. senegalensis plant (mother plant) and the M. heterophylla seeds was performed using our previously established methods [27]. In short, dried plant material was cut to pieces and grinded to dust using liquid nitrogen. The material was submerged with a mixture of ethanol:trichlormethane (80:20, v:v) and ultrasonicated for 15 min under chilled conditions. The extract was filtered, and the residue was re-extracted under identical conditions (repeated thrice). The combined extracts were pooled and evaporated with a rotary evaporator (Laborota 4001, Heidolph, Schwabach, Germany) at 40 °C water bath temperature at 100 rpm. The concentrated extract was dissolved in ethanol and measured by HPLC-HRMS using previously reported methods [28–29].
Fig. 1. Structure of maytansine.
cryptic endophytes [28]. On one hand, these results lead to the question whether different, parallel maytansine biosynthetic pathways (co-)evolved in different Celastraceae plants vis-à-vis direct or partial contribution of host plantassociated endophytic microbiome. One the other hand, given the antimicrobial and antineoplastic action of maytansine, a broader question with regard to maytansine being plausibly used as a chemical defense compound emerges: what is the ecological role of maytansine? In order to answer this open question, we have now started investigating broad spectra of both Putterlickia and Maytenus species from different geographical locations having diverse environmental conditions as well as from various botanical gardens in Germany where plants are maintained in a controlled environment. For example, we recently prospected Putterlickia pyracantha from South Africa and studied the occurrence and spatial distribution of maytansinoids in situ in different tissues using MALDI-imaging-HRMS [29]. In the present study, we have further focused on elucidating the ecological relevance of maytansine in planta particularly as a chemical defense compound. Herein we report the occurrence and spatial distribution of maytansine in a Maytenus senegalensis plant obtained from the Botanic Garden and Botanical Museum in Berlin, not only the mother plant, but also the seeds obtained from the plant, through the germination of seeds, and finally the establishment of seedlings (daughter plant). We used MALDI-imaging-HRMS, which is an effective and well-known tool for in situ detection and mapping of secondary metabolites [30], including for seeds [31–34]. The obtained results are discussed within the scope of the role of maytansine in providing chemical protection to seeds during the germination process and establishment of seedlings.
2.3. Sample preparation for MALDI-imaging-HRMS MALDI-imaging-HRMS was performed using previously published methods and instruments [29], under slightly optimized conditions. Briefly, fresh plant material (e.g., seeds, different tissues of seedlings) was cut to pieces, embedded in cellulose (5%), fixed with an OCT tissue freezing medium, and the tissues were cut to thin sections in a Thermo Scientific HM550 cryostat (35–45 μm). The cryostat had a chamber temperature of − 20 °C and the sample target was set to −15 °C. The slices were transferred onto a glass slide and fixed with thin 25 μm adhesive tape. DHB matrix solution (30 mg/mL DHB in H2O:acetone 1:1 (v:v) + 0.1% formic acid) was deposited on the samples with a SMALDI Prep spray device (TransMIT GmbH, Germany). The samples were sprayed with a matrix flow rate of 15 μL/min, nitrogen gas flow rate of 3 L/min and platform revolution speed of 130 rpm for 30 min. Before spraying, the samples were documented with the optical microscope Leica S8AP0 (Leica Microsystems GmbH, Germany) and/ or the digital microscope VHX-5000 (Keyence Deutschland GMBH, Germany), respectively. The region of interest was marked in order to relocate it again under measurement conditions.
2. Experimental 2.4. Phloroglucinol/HCl staining 2.1. Plant material For a better visualization and determination of different seedling tissues, we performed phloroglucinol/HCl staining of the transverse and longitudinal sections, as required. 2% phloroglucinol (1,3,5-trihydroxybenzene; Sigma-Aldrich) was dissolved in 95% ethanol and mixed with concentrated HCl (1:1, v:v). The sections were incubated with this solution for approximately 5 min and thereafter, shortly washed with ethanol and water.
A living Maytenus senegalensis plant (accession number YE-0-B2290101; grown from seeds originating from the Republic of Yemen; Jaijah Prov., vicinity of Kohlan, 2100–220 m, 1.6.2001, leg. Naumann s.n.) was obtained from the Botanic Garden and Botanical Museum (BGBM) in Berlin, Germany in July 2015. The plant was maintained in INFU in controlled-conditions according to the instructions of the BGBM, and was investigated for the presence of maytansine both by HPLC-HRMS (all tissues) and MALDI-imaging-HRMS (stem). In April 2016, the plant reached the seeding stage, and bore approximately 25 seeds. Some seeds were directly investigated by MALDI-imaging-HRMS, and others were planted in soil in small pots and maintained under controlled conditions in the INFU greenhouse. Under these conditions, the seeds germinated and by September 2016, the first seedlings could be obtained. The seedlings were replanted into bigger pots to obtain the daughter plants and investigated by MALDI-imaging-HRMS. In March 2017, we received another M. senegalensis plant (grown from the same seeds) from the BGBM, which was already seeding. We also investigated
2.5. MALDI-imaging-HRMS The IMS (imaging mass spectrometry) analyses were performed with an atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization source (AP-SMALDI) (TransMIT GmbH, Germany) coupled to a Q Exactive high-resolution mass spectrometer (Thermo Scientific Inc., Bremen, Germany). A 60 Hz pulsed N2 laser MNL 100 series (LTB Lasertechnik GmbH, Germany) generated the UV beam at 337.1 nm. Scan resolution was set concomitant to the sample size and measured in positive full scan ion mode at m/z 200–1000 mass 52
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Fig. 2. Schematic representation of the general workflow including MALDI-imaging-HRMS sample preparation steps.
next generation of daughter plants [6–10]. It was noteworthy that seeds of a M. senegalensis plant that did not produce maytansine, was found to produce maytansine. It would seem that the onset of seeding stage of the mother plant could serve as an internal trigger for initiation of maytansine production in the seeds. The absence of maytansine in the mother plant as well as the typically documented disjoint occurrence and detectability of maytansine in plants support this observation [19,23]. The non-detectability of maytansine in M. senegalensis tissues of the mother plant before and after the seeding stage further confirmed that maytansine is biosynthesized inside the seeds and is not transported into the seeds from a different tissue (e.g., stem) of the mother plant. More recently, we obtained another M. senegalensis plant (grown from the same seeds) that possessed freshly grown seeds. As expected, we detected maytansine in the seeds by MALDI-imaging-HRMS (Fig. S3, Supporting information), even though the mother plant was devoid of this compound. Furthermore, in order to verify whether maytansine is ecologically primed as a chemical defense compound in other species of Maytenus plant seeds, we recently bioprospected and analyzed M. heterophylla seeds from South Africa. It was revealed that the seeds not only contained maytansine, but also maytanprine and maytanbutine. The presence and spatial distribution of these maytansinoids was inspected by MALDI-imaging-HRMS, and their identity was further confirmed by extraction and HPLC-HRMS2 measurements (Figs. S4 and S5, Supporting information). Taken together, these results lend evidence to the fact that maytansine's ecological role as a chemical defense compound might not be restricted to selected Maytenus species, but a conceivable general phenomenon for Celastraceae plants.
range with an internal lock mass correction (2DHB-2H2O + H at m/z 273.03936). Measurements were generated with a mass resolution of 140.000 @ m/z 200, S-lens level 65, spray voltage of 2.0 kV, and an injection time of 300 ms. For data processing and mapping of mass pixels corresponding to the target compound(s), the software package ImageQuest (v. 1.1.0; Thermo Fisher Scientific, Germany) was used. Ion image generation was done with a bin width of ± 2.0 ppm and false colors were attached to the mass pixels. 3. Results and discussion 3.1. Investigation of the M. senegalensis mother plant The general workflow of this study is schematically represented in Fig. 2, including the different sample preparation steps for the MALDIimaging-HRMS measurements. Different tissues of the living mother plant (viz. root, stem, and leaf) were extracted individually and analyzed by HPLC-HRMS for all known maytansinoids using our previously established methods [28–29]. Interestingly, neither maytansine nor any of its structural analogs could be detected in any plant tissue. 3.2. MALDI-imaging-HRMS of seeds during germination The investigation of the seeds by MALDI-imaging-HRMS revealed the presence of maytansine inside the seeds (Fig. 3), even though the mother plant was devoid of maytansine in any tissue. Further measurements of cross-sections of the seed tissues such as the endosperm and cotyledon were performed, which are shown in the Supporting information (Fig. S1). We detected all monoisotopic masses of the adduct ions of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503) with consistent ion images, thereby confirming the accurate detection of the compound. Maytansine was found to be distributed in the cotyledons and the endosperm with an increased accumulation towards the seed coat. This pattern of accumulation at the outer area of the endosperm lends evidence that the seeds of M. senegalensis are ecologically primed to utilize maytansine as chemical defense compound against predators and/or pathogens. This would further ensure viability of the seeds and successful germination of the seeds for development of seedlings leading to the
3.3. MALDI-imaging-HRMS of seedlings after germination of seeds Some of the planted seeds germinated and generated seedlings. We investigated different parts of the seedlings, such as the epicotyl, hypocotyl, radicle, cotyledon, and foliage leaf by MALDI-imagingHRMS (Fig. 4). The sections shown in Fig. 4 are the transverse and longitudinal cuttings with a thickness of 35–45 μm. For better visualization of the anatomical structures and morphological features (e.g., the vascular bundle), we performed a phloroglucinol/HCL staining in parallel. In the hypocotyl, maytansine was found to be localized in the cortex 53
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Fig. 3. Localization of maytansine in M. senegalensis seeds by MALDI-imaging-HRMS. Optical image of transverse section along with overlaid ion images of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503; ± 2 ppm).
microorganisms [35–36]. From the chemical ecological point-of-view [37], the present results underscore the role of maytansine as chemical defense compound, providing maytansine-containing seeds and seedlings an evolutionary and ecological benefit compared to those devoid of maytansine.
and not in the vascular bundle (Fig. 4A). The adduct ions for maytansine showed consistent ion images. The potassium adduct was detected with highest intensities and was therefore used for the other ion images, in accordance with previously established methods [27,29]. Furthermore, in order to ensure curtailing any false-positive data, we re-confirmed the identity of maytansine by its characteristic pattern as the chlorinated molecule from the MALDI-HRMS spectra of [M + Na]+ and [M + K]+ adduct ions (Fig. S2, Supporting information). In the epicotyl, the maytansine was also detected in the cortex between the endodermis and the epidermis (Fig. 4B), but in lower intensities compared to that in the radicle and hypocotyl. We also investigated the foliage leaves and the cotyledons, but could not detect maytansine (< LOD). We engendered different cuttings for the investigation of the radicle. Transverse sections (Fig. 4C) as well as longitudinal sections (Fig. 4D) showed the presence of maytansine in the cortex area of the radicle, close to the epidermis. The surface of the roots was also measured by MALDI-imaging-HRMS. However, we could not detect maytansine on the surface of the epidermis of the roots (rhizodermis). In the emerging seedlings, maytansine was always detected in the cortex of the maytansine-containing tissues (i.e., the radicle, hypocotyl, and epicotyl), directly beneath the epidermis. It is compelling that maytansine provides chemical protection to the seedlings, thereby ensuring the sustainability of the next generation of daughter plants. Admittedly, whether maytansinoids are biosynthesized in the seedlings or are translocated from the seeds during the process of germination remains an open question that needs to be addressed in the future. Remarkably, the highest intensities of maytansine were found in the radicle and the hypocotyl, the two tissues that remain at the direct vicinity of soil. The rhizosphere area is well known as a “hot spot” for
4. Conclusion Since the discovery of maytansine in 1972 by the group of Kupchan [17], several studies have shown the sporadic and disjoint occurrence of maytansine in various plants. Although the plausible ecological relevance of maytansine production could be predicted, a proof-ofconcept study remained elusive so far. After more than four decades of discovery of this important compound, the present study for the first time to the best of our knowledge, provides insights into the ecospecific and/or tissue-specific production and in situ spatial/temporal distribution of maytansine in M. senegalensis plant from a mother plant through its seeds, finally to the daughter seedlings. Notably, this cytotoxic agent could be detected and visualized by MALDI-imagingHRMS in the seed endosperm typically accumulating towards the seed coats, as well as in the cortex area of seedling tissues, especially in the hypocotyl and radicle. Our results corroborate earlier studies on a plethora of other plant species where selected bioactive compounds protect the seeds and seedlings through the germination stage [6–10] against invading plant pathogens like viruses, bacteria, and fungi, as well as insects and herbivores. This, therefore, also justifies the “costs” of production of the respective secondary metabolites. Taken together, the present results lend a scientific handle on how maytansine-containing seeds and seedlings might have an evolutionary advantage of survival. Strikingly, even for mother plants where 54
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Fig. 4. Investigation of different tissues of M. senegalensis seedling by MALDI-imaging-HRMS along with cross-sections stained with phloroglucinol/HCL for identification of anatomical structures. Transverse sections of hypocotyl [spatial resolution: 30 μm; scan area: 1740 × 1800 μm (A)], epicotyl [spatial resolution: 20 μm; scan area: 1340 × 1280 μm (B)], radicle [spatial resolution: 30 μm; scan area: 1770 × 2220 μm (C)], and longitudinal section of radicle [spatial resolution: 25 μm; scan area: 5575 × 1475 μm (D)]. Localization of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503; ± 2 ppm).
Conflict of interest
maytansine is not present, their seeds and seedling stages can contain maytansine. This emphasizes the importance of studying Celastraceae plants closely for the presence of cryptic biosynthetic pathways for the production of maytansine. Additionally, given the mode of action of maytansine, it is desirable to study how plants, their seeds, and even associated microbiota have coevolved the means to resist being debilitated by the compound. For instance, earlier studies have unearthed the survival strategies and resistance mechanisms evolved in plants and associated endophytes that are able to produce bioactive (toxic) compounds, such as the anticancer compound camptothecin [38]. More importantly, it is desirable to unravel the chemical and/or molecular triggers responsible for tissue-specific “switch-on and -off” of maytansine biosynthesis, with precise emphasis on the plant tissueassociated microbiome. Further detailed studies along these lines are currently underway.
The authors do not have any conflict of interests.
Acknowledgements We are grateful to the Botanic Garden and Botanical Museum (BGBM) in Berlin, Germany, particularly Mr. Thomas Dürbye, Ms. Birgit Nordt and Dr. Nils Köster, for providing us with living Maytenus senegalensis plants. We thankfully appreciate the assistance of Mr. Ulrich Feiter of Parceval Pty. Ltd., Wellington, South Africa, in prospecting Maytenus heterophylla seeds from South Africa. The Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia, Germany, and the German Research Foundation (DFG) are thankfully acknowledged for granting a highresolution mass spectrometer and the MALDI imaging high-resolution 55
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Spatial profiling of maytansine during the germination process of Maytenus senegalensis seeds
MARK
Dennis Eckelmann, Souvik Kusari⁎, Michael Spiteller⁎ Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords: Maytansine MALDI-imaging-HRMS Seeds Seedlings Chemical defense Maytenus senegalensis
The ecological role of maytansine, an important antineoplastic and antimicrobial compound with high cytotoxicity, particularly as a chemical defense compound has remained elusive since its discovery in the 1970s in Maytenus and Putterlickia plants. In the present study, we have used MALDI-imaging-HRMS to visualize the occurrence as well as spatial and temporal distribution of maytansine in a Maytenus senegalensis plant, seeds obtained from the mother plant during seeding stage, through the germination of the seeds, and finally up to the establishment of seedlings (or daughter plants). Although the mother plant was devoid of maytansine, the bioactive compound was found to be distributed in the cotyledons and the endosperm of the seeds with an augmented accretion towards the seed coat. Furthermore, maytansine was always detected in the emerging seedlings, particularly the cortex encompassing the radicle, hypocotyl, and epicotyl. The typical pattern of accumulation of maytansine not only in the seeds but also during germination provides a proof-of-concept that M. senegalensis is ecologically primed to trigger the production of maytansine in vulnerable tissues such as seeds during plant reproduction. By utilizing maytansine as chemical defense compound against predators and/or pathogens, the plant can ensure viability of the seeds and successful germination, thus leading to the next generation of daughter plants.
1. Introduction In every ecological niche, plants coexist with a plethora of macroand microorganisms. The so-called ‘mutualistic links’ connect plants with associated microorganisms (such as endophytes and invading pathogens) and predators (such as insects and herbivores) with defined costs and fitness [1]. Concomitantly, plants coevolve physical, chemical and molecular means of defense against predators and pathogens, which are crucial not only for their existence but also for delivering vital eco-specific functions [2–5]. Notably, plants are ecologically primed to protect their seeds in order to ensure their continued existence through their offspring. This is emphasized by the fact that seed viability and germination is typically influenced not only by abiotic environmental factors but also by biotic stressors such as predators and pathogens [6–10]. In addition to structural or physical barriers, seeds often contain a repertoire of chemical defense compounds to impede invading predators and/or pathogens. For example, seeds of Phaseolus vulgaris have been shown to contain vulgarinin, an antifungal peptide highly active against fungal phytopathogens Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola, and
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Botrytis cinerea [11]. In another study, Phaseolus mungo (syn. Vigna mungo) seeds have been reported to contain an antifungal and antibacterial lysozyme [12]. Maytansine (Fig. 1) is an important anticancer, antimicrobial, and cytotoxic compound [13–16], which was first isolated by Kupchan and coworkers from Maytenus and Putterlickia plants (Celastraceae) in the 1970s [17–18]. Owing to its remarkably bioactivity, maytansine garnered immense attention of scientists worldwide, particularly in the prospection of different Celastraceae plants from diverse ecological niches [19]. These intensive efforts led to the isolation of maytansine along with its derivatives sporadically from different plants [20–23]. Additionally, some maytansine analogs were also found in other unrelated organisms such as mosses [24–25] as well as the bacteria Actinosynnema pretiosum [26]. Recently, it was established by our group that in Putterlickia verrucosa and Putterlickia retrospinosa plants, maytansine is actually biosynthesized by eco-specific root-associated endophytic bacterial community [27]. More recently, we demonstrated that the biosynthesis of maytansine in Maytenus serrata prospected from Cameroon is shared between the endophytic bacterial community colonizing the stem and the host plant containing non-culturable
Corresponding authors. E-mail addresses: [email protected] (S. Kusari), [email protected] (M. Spiteller).
http://dx.doi.org/10.1016/j.fitote.2017.03.014 Received 24 February 2017; Received in revised form 27 March 2017; Accepted 30 March 2017 Available online 03 April 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.
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this plant along with its seeds by MALDI-imaging-HRMS. In addition, in order to explore whether the spatial distribution of maytansine in M. senegalensis seeds might be representative in other Maytenus species, we bioprospected Maytenus heterophylla seeds from a mature plant in Grahamstown (S33°14,026′; E26°24,817′) in South Africa in February 2017. The prospecting of M. heterophylla seeds was realized with the help of Mr. Ulrich Feiter, Parceval Pty. Ltd., Wellington, South Africa. We analyzed the crude extract of the seeds by HPLC-HRMS and seed sections by MALDI-imaging-HRMS. 2.2. Plant extraction and HPLC-HRMS The extraction of different plant tissues (root, twig, leaf) of the M. senegalensis plant (mother plant) and the M. heterophylla seeds was performed using our previously established methods [27]. In short, dried plant material was cut to pieces and grinded to dust using liquid nitrogen. The material was submerged with a mixture of ethanol:trichlormethane (80:20, v:v) and ultrasonicated for 15 min under chilled conditions. The extract was filtered, and the residue was re-extracted under identical conditions (repeated thrice). The combined extracts were pooled and evaporated with a rotary evaporator (Laborota 4001, Heidolph, Schwabach, Germany) at 40 °C water bath temperature at 100 rpm. The concentrated extract was dissolved in ethanol and measured by HPLC-HRMS using previously reported methods [28–29].
Fig. 1. Structure of maytansine.
cryptic endophytes [28]. On one hand, these results lead to the question whether different, parallel maytansine biosynthetic pathways (co-)evolved in different Celastraceae plants vis-à-vis direct or partial contribution of host plantassociated endophytic microbiome. One the other hand, given the antimicrobial and antineoplastic action of maytansine, a broader question with regard to maytansine being plausibly used as a chemical defense compound emerges: what is the ecological role of maytansine? In order to answer this open question, we have now started investigating broad spectra of both Putterlickia and Maytenus species from different geographical locations having diverse environmental conditions as well as from various botanical gardens in Germany where plants are maintained in a controlled environment. For example, we recently prospected Putterlickia pyracantha from South Africa and studied the occurrence and spatial distribution of maytansinoids in situ in different tissues using MALDI-imaging-HRMS [29]. In the present study, we have further focused on elucidating the ecological relevance of maytansine in planta particularly as a chemical defense compound. Herein we report the occurrence and spatial distribution of maytansine in a Maytenus senegalensis plant obtained from the Botanic Garden and Botanical Museum in Berlin, not only the mother plant, but also the seeds obtained from the plant, through the germination of seeds, and finally the establishment of seedlings (daughter plant). We used MALDI-imaging-HRMS, which is an effective and well-known tool for in situ detection and mapping of secondary metabolites [30], including for seeds [31–34]. The obtained results are discussed within the scope of the role of maytansine in providing chemical protection to seeds during the germination process and establishment of seedlings.
2.3. Sample preparation for MALDI-imaging-HRMS MALDI-imaging-HRMS was performed using previously published methods and instruments [29], under slightly optimized conditions. Briefly, fresh plant material (e.g., seeds, different tissues of seedlings) was cut to pieces, embedded in cellulose (5%), fixed with an OCT tissue freezing medium, and the tissues were cut to thin sections in a Thermo Scientific HM550 cryostat (35–45 μm). The cryostat had a chamber temperature of − 20 °C and the sample target was set to −15 °C. The slices were transferred onto a glass slide and fixed with thin 25 μm adhesive tape. DHB matrix solution (30 mg/mL DHB in H2O:acetone 1:1 (v:v) + 0.1% formic acid) was deposited on the samples with a SMALDI Prep spray device (TransMIT GmbH, Germany). The samples were sprayed with a matrix flow rate of 15 μL/min, nitrogen gas flow rate of 3 L/min and platform revolution speed of 130 rpm for 30 min. Before spraying, the samples were documented with the optical microscope Leica S8AP0 (Leica Microsystems GmbH, Germany) and/ or the digital microscope VHX-5000 (Keyence Deutschland GMBH, Germany), respectively. The region of interest was marked in order to relocate it again under measurement conditions.
2. Experimental 2.4. Phloroglucinol/HCl staining 2.1. Plant material For a better visualization and determination of different seedling tissues, we performed phloroglucinol/HCl staining of the transverse and longitudinal sections, as required. 2% phloroglucinol (1,3,5-trihydroxybenzene; Sigma-Aldrich) was dissolved in 95% ethanol and mixed with concentrated HCl (1:1, v:v). The sections were incubated with this solution for approximately 5 min and thereafter, shortly washed with ethanol and water.
A living Maytenus senegalensis plant (accession number YE-0-B2290101; grown from seeds originating from the Republic of Yemen; Jaijah Prov., vicinity of Kohlan, 2100–220 m, 1.6.2001, leg. Naumann s.n.) was obtained from the Botanic Garden and Botanical Museum (BGBM) in Berlin, Germany in July 2015. The plant was maintained in INFU in controlled-conditions according to the instructions of the BGBM, and was investigated for the presence of maytansine both by HPLC-HRMS (all tissues) and MALDI-imaging-HRMS (stem). In April 2016, the plant reached the seeding stage, and bore approximately 25 seeds. Some seeds were directly investigated by MALDI-imaging-HRMS, and others were planted in soil in small pots and maintained under controlled conditions in the INFU greenhouse. Under these conditions, the seeds germinated and by September 2016, the first seedlings could be obtained. The seedlings were replanted into bigger pots to obtain the daughter plants and investigated by MALDI-imaging-HRMS. In March 2017, we received another M. senegalensis plant (grown from the same seeds) from the BGBM, which was already seeding. We also investigated
2.5. MALDI-imaging-HRMS The IMS (imaging mass spectrometry) analyses were performed with an atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization source (AP-SMALDI) (TransMIT GmbH, Germany) coupled to a Q Exactive high-resolution mass spectrometer (Thermo Scientific Inc., Bremen, Germany). A 60 Hz pulsed N2 laser MNL 100 series (LTB Lasertechnik GmbH, Germany) generated the UV beam at 337.1 nm. Scan resolution was set concomitant to the sample size and measured in positive full scan ion mode at m/z 200–1000 mass 52
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Fig. 2. Schematic representation of the general workflow including MALDI-imaging-HRMS sample preparation steps.
next generation of daughter plants [6–10]. It was noteworthy that seeds of a M. senegalensis plant that did not produce maytansine, was found to produce maytansine. It would seem that the onset of seeding stage of the mother plant could serve as an internal trigger for initiation of maytansine production in the seeds. The absence of maytansine in the mother plant as well as the typically documented disjoint occurrence and detectability of maytansine in plants support this observation [19,23]. The non-detectability of maytansine in M. senegalensis tissues of the mother plant before and after the seeding stage further confirmed that maytansine is biosynthesized inside the seeds and is not transported into the seeds from a different tissue (e.g., stem) of the mother plant. More recently, we obtained another M. senegalensis plant (grown from the same seeds) that possessed freshly grown seeds. As expected, we detected maytansine in the seeds by MALDI-imaging-HRMS (Fig. S3, Supporting information), even though the mother plant was devoid of this compound. Furthermore, in order to verify whether maytansine is ecologically primed as a chemical defense compound in other species of Maytenus plant seeds, we recently bioprospected and analyzed M. heterophylla seeds from South Africa. It was revealed that the seeds not only contained maytansine, but also maytanprine and maytanbutine. The presence and spatial distribution of these maytansinoids was inspected by MALDI-imaging-HRMS, and their identity was further confirmed by extraction and HPLC-HRMS2 measurements (Figs. S4 and S5, Supporting information). Taken together, these results lend evidence to the fact that maytansine's ecological role as a chemical defense compound might not be restricted to selected Maytenus species, but a conceivable general phenomenon for Celastraceae plants.
range with an internal lock mass correction (2DHB-2H2O + H at m/z 273.03936). Measurements were generated with a mass resolution of 140.000 @ m/z 200, S-lens level 65, spray voltage of 2.0 kV, and an injection time of 300 ms. For data processing and mapping of mass pixels corresponding to the target compound(s), the software package ImageQuest (v. 1.1.0; Thermo Fisher Scientific, Germany) was used. Ion image generation was done with a bin width of ± 2.0 ppm and false colors were attached to the mass pixels. 3. Results and discussion 3.1. Investigation of the M. senegalensis mother plant The general workflow of this study is schematically represented in Fig. 2, including the different sample preparation steps for the MALDIimaging-HRMS measurements. Different tissues of the living mother plant (viz. root, stem, and leaf) were extracted individually and analyzed by HPLC-HRMS for all known maytansinoids using our previously established methods [28–29]. Interestingly, neither maytansine nor any of its structural analogs could be detected in any plant tissue. 3.2. MALDI-imaging-HRMS of seeds during germination The investigation of the seeds by MALDI-imaging-HRMS revealed the presence of maytansine inside the seeds (Fig. 3), even though the mother plant was devoid of maytansine in any tissue. Further measurements of cross-sections of the seed tissues such as the endosperm and cotyledon were performed, which are shown in the Supporting information (Fig. S1). We detected all monoisotopic masses of the adduct ions of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503) with consistent ion images, thereby confirming the accurate detection of the compound. Maytansine was found to be distributed in the cotyledons and the endosperm with an increased accumulation towards the seed coat. This pattern of accumulation at the outer area of the endosperm lends evidence that the seeds of M. senegalensis are ecologically primed to utilize maytansine as chemical defense compound against predators and/or pathogens. This would further ensure viability of the seeds and successful germination of the seeds for development of seedlings leading to the
3.3. MALDI-imaging-HRMS of seedlings after germination of seeds Some of the planted seeds germinated and generated seedlings. We investigated different parts of the seedlings, such as the epicotyl, hypocotyl, radicle, cotyledon, and foliage leaf by MALDI-imagingHRMS (Fig. 4). The sections shown in Fig. 4 are the transverse and longitudinal cuttings with a thickness of 35–45 μm. For better visualization of the anatomical structures and morphological features (e.g., the vascular bundle), we performed a phloroglucinol/HCL staining in parallel. In the hypocotyl, maytansine was found to be localized in the cortex 53
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Fig. 3. Localization of maytansine in M. senegalensis seeds by MALDI-imaging-HRMS. Optical image of transverse section along with overlaid ion images of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503; ± 2 ppm).
microorganisms [35–36]. From the chemical ecological point-of-view [37], the present results underscore the role of maytansine as chemical defense compound, providing maytansine-containing seeds and seedlings an evolutionary and ecological benefit compared to those devoid of maytansine.
and not in the vascular bundle (Fig. 4A). The adduct ions for maytansine showed consistent ion images. The potassium adduct was detected with highest intensities and was therefore used for the other ion images, in accordance with previously established methods [27,29]. Furthermore, in order to ensure curtailing any false-positive data, we re-confirmed the identity of maytansine by its characteristic pattern as the chlorinated molecule from the MALDI-HRMS spectra of [M + Na]+ and [M + K]+ adduct ions (Fig. S2, Supporting information). In the epicotyl, the maytansine was also detected in the cortex between the endodermis and the epidermis (Fig. 4B), but in lower intensities compared to that in the radicle and hypocotyl. We also investigated the foliage leaves and the cotyledons, but could not detect maytansine (< LOD). We engendered different cuttings for the investigation of the radicle. Transverse sections (Fig. 4C) as well as longitudinal sections (Fig. 4D) showed the presence of maytansine in the cortex area of the radicle, close to the epidermis. The surface of the roots was also measured by MALDI-imaging-HRMS. However, we could not detect maytansine on the surface of the epidermis of the roots (rhizodermis). In the emerging seedlings, maytansine was always detected in the cortex of the maytansine-containing tissues (i.e., the radicle, hypocotyl, and epicotyl), directly beneath the epidermis. It is compelling that maytansine provides chemical protection to the seedlings, thereby ensuring the sustainability of the next generation of daughter plants. Admittedly, whether maytansinoids are biosynthesized in the seedlings or are translocated from the seeds during the process of germination remains an open question that needs to be addressed in the future. Remarkably, the highest intensities of maytansine were found in the radicle and the hypocotyl, the two tissues that remain at the direct vicinity of soil. The rhizosphere area is well known as a “hot spot” for
4. Conclusion Since the discovery of maytansine in 1972 by the group of Kupchan [17], several studies have shown the sporadic and disjoint occurrence of maytansine in various plants. Although the plausible ecological relevance of maytansine production could be predicted, a proof-ofconcept study remained elusive so far. After more than four decades of discovery of this important compound, the present study for the first time to the best of our knowledge, provides insights into the ecospecific and/or tissue-specific production and in situ spatial/temporal distribution of maytansine in M. senegalensis plant from a mother plant through its seeds, finally to the daughter seedlings. Notably, this cytotoxic agent could be detected and visualized by MALDI-imagingHRMS in the seed endosperm typically accumulating towards the seed coats, as well as in the cortex area of seedling tissues, especially in the hypocotyl and radicle. Our results corroborate earlier studies on a plethora of other plant species where selected bioactive compounds protect the seeds and seedlings through the germination stage [6–10] against invading plant pathogens like viruses, bacteria, and fungi, as well as insects and herbivores. This, therefore, also justifies the “costs” of production of the respective secondary metabolites. Taken together, the present results lend a scientific handle on how maytansine-containing seeds and seedlings might have an evolutionary advantage of survival. Strikingly, even for mother plants where 54
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Fig. 4. Investigation of different tissues of M. senegalensis seedling by MALDI-imaging-HRMS along with cross-sections stained with phloroglucinol/HCL for identification of anatomical structures. Transverse sections of hypocotyl [spatial resolution: 30 μm; scan area: 1740 × 1800 μm (A)], epicotyl [spatial resolution: 20 μm; scan area: 1340 × 1280 μm (B)], radicle [spatial resolution: 30 μm; scan area: 1770 × 2220 μm (C)], and longitudinal section of radicle [spatial resolution: 25 μm; scan area: 5575 × 1475 μm (D)]. Localization of maytansine ([M + H]+ m/z 692.2944; [M + Na]+ m/z 714.2764; [M + K]+ m/z 730.2503; ± 2 ppm).
Conflict of interest
maytansine is not present, their seeds and seedling stages can contain maytansine. This emphasizes the importance of studying Celastraceae plants closely for the presence of cryptic biosynthetic pathways for the production of maytansine. Additionally, given the mode of action of maytansine, it is desirable to study how plants, their seeds, and even associated microbiota have coevolved the means to resist being debilitated by the compound. For instance, earlier studies have unearthed the survival strategies and resistance mechanisms evolved in plants and associated endophytes that are able to produce bioactive (toxic) compounds, such as the anticancer compound camptothecin [38]. More importantly, it is desirable to unravel the chemical and/or molecular triggers responsible for tissue-specific “switch-on and -off” of maytansine biosynthesis, with precise emphasis on the plant tissueassociated microbiome. Further detailed studies along these lines are currently underway.
The authors do not have any conflict of interests.
Acknowledgements We are grateful to the Botanic Garden and Botanical Museum (BGBM) in Berlin, Germany, particularly Mr. Thomas Dürbye, Ms. Birgit Nordt and Dr. Nils Köster, for providing us with living Maytenus senegalensis plants. We thankfully appreciate the assistance of Mr. Ulrich Feiter of Parceval Pty. Ltd., Wellington, South Africa, in prospecting Maytenus heterophylla seeds from South Africa. The Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia, Germany, and the German Research Foundation (DFG) are thankfully acknowledged for granting a highresolution mass spectrometer and the MALDI imaging high-resolution 55
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