Histochemical detection of ace-togenins and storage moleculesin the endosperm of Annonamacroprophyllata Donn Sm.seedsG. Laguna Hernández,1A.E. Brechú Franco,1I. De-la-Cruz-Chacón,2A.R. González-Esquinca21Faculty of Sciences, NationalAutonomous University of Mexico,Coyoacán, Mexico2Institute of Biological Sciences,University of Sciences and Arts of Chiapas, Tuxtla Gtz., Chiapas, Mexico

Abstract

Acetogenins (ACGs) are bioactive com-pounds with cytotoxic properties in differentcell lines. They are antitumoural, antiparasitic,antimalarial, insecticidal, antimicrobial, anti-fungal and antibacterial. These secondarymetabolites function in plant defence and arefound in specific organelles and specific cells,thereby preventing toxicity to the plant itselfand permitting site-specific defence. The aimof this work was to histochemically determinethe in situ localisation of ACGs in theendosperm of Annona macroprophyllata seedsusing Kedde’s reagent. Additionally, the co-localisation of ACGs with other storage mole-cules was analysed. The seeds were analysedafter 6 and 10 days of imbibition, when 1 or 2cm of the radicle had emerged and metabolismwas fully established. The seeds were thentransversally cut in half at the midline andprocessed using different histological and his-tochemical techniques. Positive reactions withKedde’s reagent were only observed in fresh,unfixed sections that were preserved in water,and staining was found only in the large cells(the idioblasts) at the periphery of theendosperm. The ACGs’ positive reaction withSudan III corroborated their lipid nature.Paraffin sections stained with Naphthol BlueBlack showed reactions in the endospermparenchyma cells and stained the proteoplastsblue, indicating that they might correspond tostorage sites for albumin-like proteins. Lugol’siodine, which is similar in chemical composi-tion to Wagner’s reagent, caused a goldenbrown reaction product in the cytoplasm of theidioblasts, which may indicate the presence ofalkaloids.

Based on these results, we propose thatKedde’s reagent is an appropriate histochemi-

cal stain for detecting ACGs in situ inidioblasts and that idioblasts store ACGs andprobably alkaloids. ACGs that are located inidioblasts found in restricted, peripheral areasof the endosperm could serve as a barrier thatprotects the seeds against insects andpathogen attack.

Introduction

Species in the Annonaceae have endosper-mic seeds with minute embryos. The ruminateendosperm occupies almost the entire volumeof the seed and contains diverse storage mole-cules, including lipids, albumin-type proteins,and polysaccharides, such as starch and galac-tomannans.1-5 These storage substances areused during embryonic development, germina-tion and seedling establishment, which occursalmost a month after the onset ofgermination.6 The endosperm of the matureseeds also contains secondary metabolites,such as alkaloids, polyphenols, essential oils,terpenes and acetogenins (ACGs), which aretypical of these plants. Chemically, ACGs areC35-C38 compounds of apparent polyketide ori-gin that possess one or two tetrahydrofuranrings and a β-lactone (either saturated orunsaturated), and they usually contain aliphat-ic regions that are variously hydroxylated, ace-toxylated, or ketonised.7-10 The chromatograph-ic separation of hexane extracts of A. macro-prophyllata seeds led to the isolation of theACGs laherradurine, cherimoline-2, rollinias-tatin-2 and isorolliniastatin-2, which corre-spond to 0.1% of the endosperm’s weight.11-13

These ACGs are pharmacologically importantbecause they are potent inhibitors of the mito-chondrial NADH:ubiquinone-oxidoreductase(complex I of the respiratory chain).Laherradurine inhibits tumour cells but doesnot affect normal cells, and it could be used inthe future as an antitumor drug.14-16 These sec-ondary metabolites function in plant defenceand occur in specific organelles and specificcells, thereby preventing toxicity to the plantitself and permitting site-specific defence.Staining with Kedde’s reagent is a well-estab-lished technique used to identify ACGs inextracts from leaves, seeds and other plantstructures by thin layer chromatography; itreacts with the β-unsaturated-γ-lactone moi-ety commonly found in annonaceousACGs.8,17,18

The aim of this work was to histochemical-ly determine the in situ localisation of ACGsin sections of A. macroprophyllata seedsusing Kedde’s reagent, as well as to analysethe co-localisation of ACGs with other storagemolecules.

Materials and MethodsPlant materials

A. macroprophyllata seeds were collected inSan Lucas, Chiapas, Mexico. The herborisedsample was collected in June 2013 and wasdeposited in the Eizi Matuda Herbarium of theUniversidad de Ciencias y Artes de Chiapas,Tuxtla Gutiérrez, Chiapas, Mexico under refer-ence voucher #354.

Germination conditionsThe seeds were germinated in Conviron

environmental chambers that were main-tained at 24-28°C with 12 h light/dark cyclesand a relative humidity of approximately 65-75%. Light was delivered by cool white ultra-high-output fluorescent lamps at an intensityof 500 mW m-2 s-1.

Growth testA total of 50 uncoated seeds were rehydrated

on a paper sheet under controlled temperatureand light conditions.19 Consistent with previousresults,20 65% of the seeds germinated, and theseedlings were selected after 10 days of imbibi-

European Journal of Histochemistry 2015; volume 59:2502

Correspondence: Guillermo Laguna Hernández,Facultad de Ciencias, Universidad NacionalAutónoma de México, Avenida Universidad 3000,Colonia Universidad Nacional Autónoma deMéxico, C.U., Coyoacán 04510, México. Tel. +52.55.56225431 – Fax: +52.55.56224828. E-mail: glagher@ciencias.unam.mx

Key words: Annona; Kedde’s reagent; acetogeninhistochemistry; storage reserves histochemistry;acetogenin idioblast.

Contributions: all authors had substantial contri-butions to the acquisition, analysis, or interpreta-tion of data for the work.

Conflict of interest: the authors declare no con-flict of interest.

Acknowledgments: the authors thank Dr. PatriciaRivas Manzano and Biol. Carlos Tonatiuh ChaviraRamírez for technical assistance with the cryo-stat sectioning in the Laboratory of Tissue andReproductive Biology, Faculty of Sciences,National Autonomous University of Mexico.

Received for publication: 29 January 2015.Accepted for publication: 5 April 2015.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright G. Laguna Hernández et al., 2015Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2015; 59:2502doi:10.4081/ejh.2015.2502

[European Journal of Histochemistry 2015; 59:2502] [page 239]

EJH_2015_03-tecnical 239-250.qxp_Hrev_master 12/10/15 10:10 Pagina 239

Non co

mmercial

use o

nly

[page 240] [European Journal of Histochemistry 2015; 59:2502]

tion, when the radicle was 1 or 2 cm in lengthand metabolism was fully established. Theseeds were then cut in half at the midline andprocessed as illustrated in Figure 1.

Seeds used for fresh sectionsSome of the seed halves were stored in water

until sectioning on a cryostat. Others werestored in 70% alcohol, and others were fixed inFAA (10% formaldehyde/50% alcohol/5% aceticacid/35% water) for 72 h or in AGF (1.5%acrolein/3% glutaraldehyde/1.5% formaldehydein 0.1 M sodium cacodylate buffer, pH 7.4)(Electron Microscopy Sciences, Hatfield, PA,USA) for 12 h. Then, the seed halves werewashed with distilled water. Some halves wereprocessed for paraffin embedding,21 and othersremained in water until sectioning.

Histochemistry on fresh sectionsThe seeds that were stored in water,

whether fixed or not, and the seeds that werestored in 70% alcohol were cut into 20-µmcross sections using a Leica CM1510S cryostatat between -14 and -18°C. Once on slides, thefresh sections were washed with distilledwater three times for 5 min per wash to elimi-nate the Tissue Tech gel. The excess waternear the section was absorbed with filterpaper, and then the appropriate reagent wasadded. Kedde’s reagent8 (10% dinitrobenzoicacid and 2 N KOH in MeOH) is specific forACGs, and a positive reaction yields apink/magenta color. Lugol’s iodine solution(0.5 g iodine and 2.0 g KI in 100 mL distilledwater) yields a dark purple color when it reactswith starch. Sudan III (0.1% in EtOH)18 yields areddish orange color in the presence of fats. Allanalyses were performed at room temperature(approximately 23°C). After the reagents wereapplied, the samples were immediatelyobserved through a microscope for ten to thirtyminutes and photographed.

Histochemistry on paraffin sectionsThe paraffin-embedded samples were cut

into 8-µm sections using a Microm HM 340 Emicrotome (Thermo Fisher Scientific, Inc.,Waltham, MA, USA), deparaffinised, rehydrat-ed, stained for 10 min with Kedde’s reagent tovisualise the ACGs, and observed under amicroscope. Other sections were rehydrated in70% EtOH and stained with Naphthol BlueBlack (1.0% in 50% EtOH) for protein analysis,and others were rehydrated in water, exposedto periodic acid (0.6%) for 1 min, and rinsedwith distilled water. Then, Schiff reagent(Sigma-Aldrich 3952016; S. Louis, MO, USA)was added, incubated for 20 min and rinsedwith distilled water. Then, the sample wasdehydrated with xylene and mounted with

resin. With this treatment, the polysaccharidesstained a fuchsia color. Johansen’s quadruplestain was added to sections that had beenrehydrated in 70% EtOH. According to

Johansen, although the mixtures are rathercomplicated, the procedure is simple; differen-tiation is automatic, and little is left to person-al judgment.21

Technical Note

Figure 1. Methods flowchart.

Figure 2. A) Kedde’s reagent positively reacts with the acetogenins in the idioblasts offresh, unfixed sections, yielding a pink/magenta color. B) Sudan III positively reacts withoil drops to yield a reddish orange color in fresh, unfixed sections. C) Kedde’s reagentdoes not react with fresh sections fixed in FAA; the idioblasts appear empty. D) NaphtholBlue Black reagent stains the cell walls and proteoplasts of paraffin sections blue, and theidioblasts appear empty. Ep, endosperm parenchyma; I, idioblasts; Od, oil drops; P, pro-teoplasts.

EJH_2015_03-tecnical 239-250.qxp_Hrev_master 12/10/15 10:10 Pagina 240

Non co

mmercial

use o

nly

[European Journal of Histochemistry 2015; 59:2502] [page 241]

MicrophotographyPhotographs and idioblast measurements

were taken with a Canon PowerShot A640digital camera coupled to a Zeiss AxiostarMicroscope with AxioVision software (CarlZeiss Imaging Systems, Jena, Germany).Microscopic observations were made with A-Plan 10x/0.25 and A-Plan 40x/0.65 Ph2 objec-tives.

Results and Discussion

Positive reactions with Kedde’s reagentwere only observed in fresh sections that wereeither unfixed or had been fixed in AGF andpreserved in water, and staining was foundonly in the large cells (56×38 µm) at theperiphery of the endosperm next to thetegmen; these cells are idioblasts (Figure 2A).The lipid nature of the ACGs was confirmed bytheir positive reaction with Sudan III in bothfresh sections and sections that had been fixedin AGF (Figure 2B). Therefore, we propose thathistochemical staining is appropriate fordetecting ACGs and that the large cells areidioblasts that store ACGs.

Due to the use of solvents such as ethanoland xylene, none of the sections that had beenfixed in FAA, stored in 70% alcohol, or embed-ded in paraffin showed staining in theidioblasts (Figure 2C). Naphthol Blue Blackstained the cell walls blue, and the idioblastcytoplasm appeared secretory and lacked anyspecial organelles; in the remainingendosperm cells, blue-stained proteoplastswere detected (Figure 2D) and might corre-spond to storage sites for albumin-type pro-teins, which have been reported to be stored inthe endosperm of A. muricata5 and A. macro-prophyllata22 seeds. Johansen’s quadruplestain turned these organelles purple (Figure3A), confirming the presence of proteinreserves.

The presence of oils,23 which act as reservesubstances in the endosperm, was evidentbased on their reactivity with Sudan III (Figure2B) and, indirectly, by the spaces observed inthe cells in the paraffin-embedded material(Figure 3B). The material fixed in AGF, whichhad been preserved in water and sectioned ona cryostat, still contained ACGs in theidioblasts. The plastids were not stained bySchiff reagent (Figure 3C), demonstrating thatthey do not contain starch; their reaction withthe crystal violet in Johansen’s quadruple stainindirectly indicates that they contain proteins(Figure 2D).

Staining with Lugol’s iodine (Figure 3D)did not indicate the presence of starch, butthe reagent generated a golden brown color in

the idioblasts in the same place as the reac-tion with Kedde’s reagent, indicating thepresence of dextrins.5 However, the contentappeared more like a drop-shaped liquid thanthe expected solid particles. This goldenbrown reaction product between the cytoplas-mic content of the idioblasts and Lugol’siodine, which is similar in chemical composi-tion to Wagner reagent,24 may indicate thepresence of alkaloids. Therefore, this reactiondeserves further histochemical study; thebiosynthesis of liriodenine, an aporphinicalkaloid, begins during the early stages ofdevelopment of seedlings of Annona macro-prophyllata Syn A. diversifolia Saff.6

ACGs are bioactive compounds with cytotox-ic properties in multiple cell lines, and theyhave antitumour, antiparasitic, antimalarial,insecticidal, antimicrobial, antifungal andantibacterial effects.15 In particular, the aceto-genin laherradurin might act as an autoinduc-er or quorum-sensing signalling molecule thataffects the expression of genes involved inbiofilm formation in Pseudomonas plecoglossi-cida J26.18 Since ACGs are located in idioblaststhat are found only in restricted areas in theperiphery of the endosperm, despite the largeamount of protein and oil reserves contained

in the endosperm of mature seeds,25 ACGs mayact as a barrier that protects seeds againstinsects and pathogens. In addition, the pres-ence of alkaloids in the idioblasts might sup-port this idea, because alkaloids are alsostrongly insect repellent and toxic to microor-ganisms.17

Villamil et al. reported amyloid granules inA. muricata endosperm.5 However, in A. macro-prophyllata, amyloids are not present becausedo not show blue or purple staining withLugol’s iodine in the endosperm cell walls, asreported by Kooiman2 for 25 species ofAnnonaceae. Some endosperm cells in A.macroprophyllata stain brown, which may cor-respond to dextrins. As described by Kooiman,2

the term amyloid refers to a group of polysac-charides with similar structures, particularlythose reported by Schleiden in 1838 (in 2) andby Vogel and Schleiden in 1839 (in 2), who firstused the term. Using Mitchell reagent, whichhas the same composition as Lugol’s iodine,Kooiman detected cellulose and hemicellulosevia blue staining in the presence of 75% sul-phuric acid. Consistent with these ideas, wesuggest that the term amyloid should nolonger be used to refer to the group of com-pounds that can be detected with Schiff

Technical Note

Figure 3. A) Johansen’s quadruple stain used on a paraffin section stains the proteoplastspurple. B) Kedde’s reagent used on a paraffin section does not react, and the idioblastsappear empty. C) Schiff reagent used on a paraffin section stains the cell walls purple, butthe proteoplasts and idioblasts do not stain. D) Lugol’s iodine used on a fresh, unfixedsection results in golden brown-colored drops in the idioblasts. Ep, endosperm parenchy-ma; I, idioblasts; P, proteoplasts.

EJH_2015_03-tecnical 239-250.qxp_Hrev_master 12/10/15 10:10 Pagina 241

Non co

mmercial

use o

nly

[page 242] [European Journal of Histochemistry 2015; 59:2502]

reagent,21 as the term is now used to refer to abeta-fibrillar protein of animal origin, the amy-loid beta-protein.26

This new histochemical method for detect-ing ACGs in situ in seed sections using Kedde’sreagent can test for the presence of these sub-stances in developing tissue or mature organs.It can also differentiate between various sub-stances in the same structures, as it occurredusing Warner reagent to detect alkaloids inidioblasts. This work histochemically demon-strates the compartmentalisation of ACGs inthe endosperm, suggesting that its aforemen-tioned characteristics are part of a protectivestrategy that allows selective defence for thesexual propagation of the species.

References

1. Corner EJH. Annonaceous seed and itsfour integuments. New Phytol 1949;48:332-64.

2. Kooiman P. On the occurrence of amyloids inplant seeds. Acta Bot Neerl 1960;9:208-19.

3. Heijden E Van der, Bouman F. Studies inAnnonaceae. Seed anatomy of the Annonagroup. Bot Jahrb Syst 1988;110:117-35.

4. Svoma E. Seed development and functionin Artabotrys hexapetalus (Annonaceae).Plant Syst Evol 1997;207:205-23.

5. Villamil H, Corchuelo G, Valencia M.Morfología, anatomía de la semilla y com-posición química del endospermo deAnnona muricata L. Agron Costar 1999;16:19-23.

6. De la Cruz-Chacón I. González-EsquincaAR. Liriodenine alkaloid in Annona diver-sifolia during early development. Nat ProdRes 2012;26:42-9.

7. Ruprecht JK, Hui YH, McLaughlin JL.Annonaceous acetogenins: a rewiew. J NatProd 1990;53:237-78.

8. Cavé A, Cortes D, Figadère B, Laurens A,Pettit GR. Acetogenins from Annonaceae.In: W. Herz, G.W. Kirby, R.E.W. Moore, W.

Steglich and C.H. Tamm (eds.) Progress inthe chemistry of organic natural products.Springer, Wien; 1997; pp. 81-288.

9. Alali FQ, Liu XX, McLaughlin JL.Annonaceus acetogenins: recent progress.J Nat Prod 1999;62:504-40.

10. Bermejo A, Figadère B, Zafra-Polo MC,Barrachina I, Estornell E, Cortes D.Acetogenins from Annonaceae: recentprogress in isolation, synthesis and mech-anisms of action. Nat Prod Rep 2005;22:269-303.

11. De la Cruz-Chacón I. AcetogeninasBioactivas de Annona diversifolia Safford.Tesis de Licenciatura. InstitutoTecnológico de Tuxtla Gutiérrez. TuxtlaGutiérrez, Chiapas; 2001.

12. Schlie-Guzmán MA, García Carranca A,González-Esquinca AR. In vitro and in vivoantiproliferative activity of laherradurinand cherimolin-2 of Annona diversifoliaSaff. Phytother Res 2009;23:1128-33.

13. Luna-Cazáres L, González-Esquinca AR.Susceptibility of bacteria and completespheroplasts of Escherichia coli,Pseudomonas aeruginosa and Salmonellatyphi to rolliniastatin-2. Nat Prod Res2010;24:1139-45.

14. Zafra-Polo MC, Figadère B, Gallardo T,Tormo R J, Cortés D. Natural acetogeninsfrom Annonaceae, sinthesis and mecha-nism of action. Phytochemistry 1998;48:1087-117.

15. De Pedro N, Cautain B, Melguizo A, CortesD, Vicente F, Genilloud O, et al. Analysis ofcytotoxic activity at short incubation timesreveals profound differences amongAnnonaceus acetogenins, inhibitors ofmitochondrial Complex I. J. Bioenerg.Biomembr 2013;45(1-2):145-152.

16. Cortes D, Moreno L, Párraga J, Galán A,Cabedo N. Nuevos fármacos inspirados enAnnonaceas. Rev Bras Frutic 2014;36:22-31.

17. Cunha MM, Nascimento FC, Santos-Pimenta LP, Boaventura MAD, Salas CE,Lopes MTP. Screening of cytitoxic activityin hexanic and ethanolic extracts of

Rollinia laurifolia. Lat Am J Pharm2009;28:234-40.

18. Parellada EA, Ferrero M, Cartagena E,Bardón A, Neske A. Laherradurin, a natu-ral stressor, stimulates QS mechanisminvolved in biofilm formation of a PAHsdegrading bacterium. Int BiodeterBiodegrad 2013;85:78-84.

19. Ministerio de Agricultura. Reglas interna-cionales para ensayos de semillas. InstitutoNacional de Semillas y plantas de Vivero.Dirección General de la Producción Agraria.Reimpreso por la SARH, México; 1976.

20. González-Esquinca AR, De la Cruz-ChacónI, Dominguez-Gutú LM, Latency and ger-mination of Annona macroprophyllataDonn. Sm (Annonaceae): the importanceof micropylar plug and seed position. Bot.Sci 2015 (In press).

21. Johansen DA. Plant microtechnique.McGraw-Hill Book Co., Inc., New York; 1940.

22. Ferreira G. Reguladores vegetais na super-acao da dormencia, balanco hormonal edegradacao de reservas em sementes deAnnona diversifolia SAFF. e A purpureaMoc. Sesse ex Dunal (Annonaceae).Thesis, UNESP Institute of Biosciences,Botucatu, Brazil; 2011.

23. Pérez-Amador MC, González-Esquinca A,García Argaez A, Bratoeff E, Labastida C.Oil composition and flavonoid profiles ofthe seeds of three Annona species. PhytonInt J Exp 1997;61:77-80.

24. Corsi G, Biasci D. Secretory structures andlocalization of alkaloids in Conium macu-latum L. (Apiaceae). Ann Botany 1998;81:157-62.

25. De la Cruz-Chacón I. Alcaloides benciliso-quinolínicos y su relación órgano-específi-ca durante las primeras fases del desarro-llo de Annona diversifolia Safford. PhDThesis, National Autonomous Universityof Mexico; 2012.

26. Kyle RA. 2001. Amyloidosis: a convolutedstory. Brit J Haematol 2001;114:529-38.

Technical Note

EJH_2015_03-tecnical 239-250.qxp_Hrev_master 12/10/15 10:10 Pagina 242

Non co

mmercial

use o

nly