The Endosperm of a Rare Tree Endemic to Chile, Gomortega Keule, Has Two Parts with Different Chemical Composition

New Zealand Journal of Botany

ISSN: 0028-825X (Print) 1175-8643 (Online) Journal homepage: http://www.tandfonline.com/loi/tnzb20

The endosperm of a rare tree endemic to Chile, Gomortega keule, has two parts with different chemical composition

Diego Muñoz-Concha, Carlos Valdivia, Patricio Peñailillo & Carlos Baeza

To cite this article: Diego Muñoz-Concha, Carlos Valdivia, Patricio Peñailillo & Carlos Baeza (2018): The endosperm of a rare tree endemic to Chile, Gomortega keule, has two parts with different chemical composition, New Zealand Journal of Botany, DOI: 10.1080/0028825X.2018.1468343 To link to this article: https://doi.org/10.1080/0028825X.2018.1468343

Published online: 10 May 2018.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tnzb20 NEW ZEALAND JOURNAL OF BOTANY https://doi.org/10.1080/0028825X.2018.1468343

SHORT COMMUNICATION The endosperm of a rare tree endemic to Chile, Gomortega keule, has two parts with different chemical composition Diego Muñoz-Conchaa, Carlos Valdiviaa, Patricio Peñailillob and Carlos Baezac aDepartamento de Ciencias Agrarias, Universidad Católica del Maule, Curicó, Chile; bInstituto de Ciencias Biológicas, Universidad de Talca, Talca, Chile; cDepartamento de Botánica, Universidad de Concepción, Concepción, Chile

ABSTRACT ARTICLE HISTORY ‘Queule’ (Gomortega keule) is an endemic and endangered Chilean Received 25 November 2017 tree belonging to the monotypic family Gomortegaceae. Inside its Accepted 19 April 2018 edible fruit the usually single seed has an oily endosperm divided ASSOCIATE EDITOR in two distinctive parts; this represents a unique organisation of Liliana Katinas storage tissue in angiosperm seeds. Since this trait has not been previously reported in literature, this study aimed to describe both KEYWORDS parts of the storage tissue and to identify the presence of lipid, Embryo; endosperm; starch and protein. Seeds were observed using the paraffin Gomortegaceae; Laurales; oil; method and handmade slices. Specific dyes were applied to seed anatomy identify chemical compounds. Oil content was quantified in both parts of the endosperm. Additionally, seed and embryo size were measured from endocarps collected from the forest floor during an 8 month period. Increased embryo size and absence of germination suggest morphological dormancy. Histological slices showed abundant and larger starch grains in the inner part of the endosperm, and lower presence of proteins compared to the outer part. In terms of oil content, the inner part of the endosperm contained 27% (w/w fresh weight), the embryo 22% and the outer part 20%. Although both parts of the storage tissue are notoriously different, both belong to the endosperm. The presence of oil, proteins and starch in the storage tissue may provide ecological advantages for seedling establishment.

Introduction Gomortega keule (Molina) Baillon, commonly named ‘queule’, belongs to the monotypic family Gomortegaceae within the order Laurales (Byng 2014). This evergreen tree is endemic to the coastal ranges of the Maule and Bío-Bío regions of central Chile (Benoit 1989; Hechenleitner et al. 2005), being a characteristic component of the Chilean Winter Rainfall-Valdivian biodiversity hotspot (Myers et al. 2000). The conservation status of the species is Endangered because of the intense replacement of its natural habitat by plantations of exotic timber trees (Espinosa 1948; Le-Quesne and Stark 2006). The edible fruit of G. keule can be consumed fresh or processed, and has been known to the local people since ancient times (Le-Quesne and Stark 2006). The excellent quality wood was used for crafting. The attractive appearance of the tree gives it an

CONTACT Diego Muñoz-Concha [email protected] © 2018 The Royal Society of New Zealand 2 D. MUÑOZ-CONCHA ET AL. ornamental potential (Albert 1924; Rodríguez et al. 2007). However, the species is not cul- tivated for human use due to its difficult propagation. The fruit of G. keule is a false drupe developed from an inferior ovary (Byng 2014). The extremely hard endocarp encloses one (or, rarely, two or three) seed(s) which has been described as oily (Muñoz 1986). The seed anatomy was reported by Doweld (2001) who refers to the storage tissue as endosperm without adding any further descriptions. However, when the seed is microdissected, two distinct parts (inner and outer) can be observed in the endosperm. Heo et al. (2004) showed a photograph of the seed where the two parts could be distinguished, without further information. This work aimed to describe the chemical composition of the storage tissue in the seed of G. keule and to characterise the presence of organic substances such as lipids, proteins and starch. Additionally, embryo size was evaluated as complementary data to this poorly known seed.

Materials and methods Plant material

Fruits and endocarps of G. keule were obtained from Los Queules National Reserve (35° 58′S, 72°40′W) and Ralbún (Forestal CELCO S.A.; 36°03′S, 72°38′W), Chile. Mature fruits were collected from the forest floor in March for microscopic observation. Endocarps were obtained from the forest floor in December to evaluate oil content. Embryo size was measured using endocarps left on the litter of the forest floor in a mesh cage, thus preventing disturbances by animals or people; 6–19 endocarps were taken every month during 10 months and fixed in FAA (40% formaldehyde, 96% ethanol, acetic acid, water; 10:50:5:35). Endocarps were opened using a bench vice and seeds were subsequently dissected. Embryo length and width were measured and a logistic regression (Thornley and Johnson 1990) was performed to evaluate size changes.

Microscopy preparations

For microscopy preparations, the procedure by Chamberlain (2007) was followed with modifications. Fixed seeds were dehydrated in a series of ethanol solutions (50%, 60%, 70%, 80% and 96%), and subsequently placed in petroleum ether, xylol and paraffin. Sample sections of 18 µm were obtained using a microtome Microm HM-35 and subsequently immersed in xylol and ethanol. After staining with safranin (red) and fast green according to D’Ambrogio (1986), the samples were mounted using Histofluid medium. Histochemical tests were performed for proteins (eosin 1%, staining red-pink; Barber et al. 1991), lipids (Sudan III 0.2%, staining orange-red; Schneider et al. 1999) and starch (iodine 1%–potassium iodide 2%, staining reddish brown or bluish; Wang et al. 1998) on handmade slices of fresh seeds obtained with a razor blade. Tissues were observed in a microscope Olympus CX31 under different magnifications. Photographs were taken with an MShot MC50 digital camera.

Oil content determination

Seeds were dissected and the embryo, inner and outer part of the storage tissue were sep- arated, weighed and thoroughly ground in a 1.5 mL centrifuge plastic tube. The tube was NEW ZEALAND JOURNAL OF BOTANY 3 centrifuged at 8000 rpm for 5 min. The two liquid phases observed were taken using a micropipette and weighed. The process was repeated with the pellet at 10,000 rpm for 7.5 min. The extraction was performed three times independently, each with 10 seeds. In order to detect oil presence, the two previously retrieved liquids phases were treated with Sudan III. For dry mass determination, tissue remains were dried after the extraction using a Binder heater at 60 °C for 48 h.

Results Embryo size

During the time the endocarp was lying on the forest floor, the length and width of the embryo increased, fitting a logistic growth equation (r2 = 0.83 for length and 0.69 for width; Figure 1). Protrusion of the radicle was not observed.

Microscopy observations

Two parts of the endosperm were clearly distinguishable in the histological preparations (Figure 2A). The outer part of the endosperm showed parenchymal cells containing small green-hyaline structures stained with fast green and consequently corresponding to protein bodies (Prego et al. 1998). Big red grains were visible in cells from the inner part of the seed. The cells from both parts got together without a distinct limit or cell arrangement (Figure 2B). The inner part was stained with iodine-potassium iodide, while the outer part showed weak and diffuse staining with the same dye. A higher abundance of large starch grains was observed in the inner part than in the outer part of the endosperm.

Figure 1. Embryo size changes in Gomortega keule. 4 D. MUÑOZ-CONCHA ET AL.

Figure 2. Storage tissue in the seed of Gomortega keule. A, General view of the seed showing the inner part (IP) and outer part (OP) of the endosperm. Scale bar = 0.5 mm; B, contact zone between the OP and IP. Scale bar = 200 µm; C, iodine-potassium iodide staining showing starch grains in the inner part. Scale bar = 30 µm; D, inner part showing big oil bodies (OB) stained with Sudan III. Scale bar = 50 µm.

Using hand preparations, the outer part showed starch grains stained with iodine-potassium iodide, oil bodies stained with Sudan III and small protein bodies stained with eosin. The inner part presented grains stained with iodine (Figure 2C) corresponding to starch or maybe to a mixture of starch and proteins (Ancíbor 1971). Big oil bodies were stained with Sudan III (Figure 2D). Regarding the relative abundance of the different particles stained in each part of the endosperm, the outer part showed an abundance of lipids (oil), proteins and the presence of starch. The inner part contained an important proportion of lipids, starch grains and a lower content of proteins (Table 1).

Oil presence and content

After grinding and centrifugation, two distinct liquid phases were distinguished in the embryo and the endosperm parts (outer and inner). The oily liquid (upper liquid

Table 1. Presence of substances detected by dyes in the outer and inner part of the endosperm of Gomortega keule. Dye Substance Outer part Inner part Safranin (microtome) Starch scarce presence of grains presence of big polyhedral grains Iodine (hand preparation) Starch grains in cell periphery grains in cell periphery Iodine (microtome) Starch presence of small grains presence of big grains Sudan III (hand preparation) Lipids presence of big bodies presence of big bodies Fast Green (microtome) Proteins presence of small grains some small grains Eosin (hand preparation) Proteins presence of small grains some small red grains NEW ZEALAND JOURNAL OF BOTANY 5 phase) was positively stained with Sudan III, confirming its lipidic nature. Oil, dry matter and water contents for each part of the endosperm and the embryo are shown in Figure 3.

Discussion Embryo size

The results suggest that embryo length increases over time (Figure 1). This effect is less evident in embryo width. The increase in size may correspond to a pre-germinative matu- ration of the embryo. This would account for the long time needed for germination in this species. The seed of G. keule does not germinate before 5 months (Donoso and Escobar 1985; Serra et al. 1986; Cabello 1987; Orellana 1996), and some authors have observed a considerably longer germination time (Maldonado 1990; Morales and Calquín 2004). The long time of embryo growth observed in the present study would fit well into the category of morphological dormancy proposed by Baskin and Baskin (2004), having a rela- tively small but differentiated embryo. However, the time required for germination in G. keule is much greater than the range defined for this category of dormancy. The large seed size and the low temperatures in the forest may explain this germination delay.

Microscopy

The presence of two distinct parts in the endosperm of G. keule is intriguing. In related families (within the order Laurales), the storage tissue in the seed, when present, corre- sponds to the endosperm. In Hernandiaceae and Lauraceae, the endospem is absent (Kimoto and Tobe 2008), although it remains present in Monimiaceae, Hortonia,

Figure 3. Mean weight (mg) of the components in the embryo and the two parts (inner and outer) of the endosperm in the seed of Gomortega keule. 6 D. MUÑOZ-CONCHA ET AL.

Siparunaceae and Atherospermataceae (Kimoto and Tobe 2001). Mature seeds of Siparu- naceae contain an oily endosperm and no perisperm (Kimoto and Tobe 2003). Mature seeds of G. keule also lack nucellar tissue (i.e. perisperm; Heo et al. 2004), as in Monimia- ceae and Hortonia (Kimoto and Tobe 2008). The detection of lipids in handmade slices, but not in paraffin-embedded samples, is explained by the use of solvents and the consequent removal of lipids during dehydration in microtome preparations. Oily bodies in the inner part of the endosperm were observed to be larger than in the outer part of the endosperm. Interestingly, the presence of oil droplets in parenchymatic cells of the endosperm of G. keule was previously reported by Heo et al. (2004). Starch grains in the seed of G. keule ranged 4–42 µm (mean 23 µm), larger than the 3– 15 µm reported in Myrtaceae species (Cortadi et al. 1996). Protein bodies (mean 47 µm) were larger than those observed in the seed of Cuphea glutinosa (8 µm, Lythraceae) by Di Santo et al. (2012). To our knowledge, there are no previous reports of the size of starch grains or protein bodies in Laurales.

Oil presence and content

The oily nature of the endosperm of G. keule was first mentioned by Reiche (1896) and, more recently, by Kimoto and Tobe (2001). In the present study, the inner part of the endosperm had 26.9% (w/w) of lipids, while the embryo had 22.5% and the outer part 19.6% (based on fresh weight). Including the seed coat, the mean oil content was 18.2%. In oil crops such as soya bean the seed oil content is typically 18% and over 40% for sunflower or peanut, respectively (Sánchez et al. 1987). This result would support a description of G. keule seeds as being oily (Chadefaud and Emberger 1960). In the family Siparunaceae, phylogenetically related to Gomortegaceae, the genus Sipar- una has an endosperm with oil droplets within cells (Kimoto and Tobe 2003). The same observation is reported for the Monimiaceae genera Monimia, Peumus and Palmeria, the latter also having an oily endosperm (Kimoto and Tobe 2008). Muñoz (1986) mentioned that the seed is edible and that it has an oily flavour, but no records of seeds used for food or for medicinal purposes have been published to support this claim (Muñoz-Concha and Garrido-Werner 2011). The presence of a variety of storage compounds (oil, protein and starch) in the seed of G. keule may constitute a strategy for seedling development. This would provide flexibility and robustness to the ecological performance of the species (Lüttge 2012).

Conclusions The embryo of G. keule undergoes a size increment during the several months it goes without germination, which would be a characteristic of morphological dormancy. Evident differences in the cell contents were observed between the two parts of the endosperm. The inner part typically bears more abundant and larger starch grains than the outer part. The outer part has a higher presence of protein bodies. Both parts contain abundant lipids, which represent 27% (w/w fresh weight) of the inner part, 22% of the embryo and 20% of the outer part of the endosperm. These observations do not consider different tissue identities for the two parts of the endosperm (e.g. perisperm) in G. keule, NEW ZEALAND JOURNAL OF BOTANY 7 despite the notable differences between them. The absence of perisperm in related families supports this interpretation.

Acknowledgements The authors wish to thank the Corporación Nacional Forestal (CONAF), especially Fernando Campos for his field support. Thanks to Forestal CELCO S.A. for field permissions. Thanks to Víctor Monzón for facilitating access to equipment, and to Andrea Garrido for her fieldwork.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding The Government of Chile—CONICYT (Comisión Nacional de Investigación Científica y Tecnoló- gica) [FONDECYT Iniciación 11110375] partly funded this research.

References Albert F. 1924. Materias primas vegetales y animales. Santiago: Sociedad Imprenta y Litografía Universo. p. 35. Ancíbor E. 1971. Estudio anatómico y morfológico de una crucífera andina en cojín: Lithodraba mendocinensis. Darwiniana. 16:519–561. Barber DL, Lott JNA, Harris DA. 1991. Practical methods for identification of rice endosperm protein bodies and fecal protein particles in light microscopy. Food Structure. 10:137–144. Baskin JM, Baskin CC. 2004. A classification system for seed dormancy. Seed Science Research. 14:1–16. Benoit I. 1989. Libro rojo de la flora terrestre de Chile. Santiago: Corporación Nacional Forestal. p. 157. Byng JW. 2014. The flowering plants handbook: a practical guide to families and genera of the world. Hertford: Plant Gateway Ltd. p. 619. Cabello A. 1987. Proyecto de protección y recuperación de especies arbóreas y arbustivas amenazadas de extinción (II Parte y final). Santiago: Corporación Nacional Forestal. p. 8. Chadefaud M, Emberger L. 1960. Traité de Botanique. Tome 2. Paris: Masson et Cie. Édituers. p. 1540. Chamberlain CJ. 2007. Methods in plant histology. 4th ed. Chicago: The University of Chicago Press. p. 328. Cortadi A, Di Sapio O, Gattuso M. 1996. Caracteres anatómicos de tres especies medicinales de la familia Myrtaceae. Acta Farmacéutica Bonaerense. 15:109–123. D’Ambrogio A. 1986. Manual de técnicas en histología vegetal. Buenos Aires: Hemisferio Sur. p. 83. Di Santo ME, Cardinali FJ, Thevenon MA. 2012. Estudio anatómico y distribución de las reservas proteicas y lipídicas en semillas de diferentes tamaños de Cuphea glutinosa (Lythraceae). Boletín de la Sociedad Argentina de Botánica. 47:341–350. Donoso C, Escobar B. 1985. Germinación de Gomortega keule (Mol.) Baillon. Bosque. 6:120–122. Doweld A. 2001. Carpology and permathology of Gomortega: systematic and evolutionary impli- cations. Acta Botánica Malacitana. 26:19–37. Espinosa M. 1948. Estudios botánicos. 2. Nomenclatura del queule. Boletín del Museo Nacional de Historia Natural. 24:52–79. Hechenleitner P, Gardner MF, Thomas PI, Echeverría C, Escobar B, Brownless P, Martínez C. 2005. Plantas amenazadas del centro-sur de Chile. Valdivia: Universidad Austral de Chile y Royal Botanic Garden of Edinburgh. p. 188. 8 D. MUÑOZ-CONCHA ET AL.

Heo K, Kimoto Y, Riveros M, Tobe H. 2004. Embryology of Gomortegaceae (Laurales): characteristics and character evolution. Journal of Plant Research. 117:221–228. Kimoto Y, Tobe H. 2001. Embryology of Laurales: a review and perspectives. Journal of Plant Research. 114:247–267. Kimoto Y, Tobe H. 2003. Embryology of Siparunaceae (Laurales): characteristics and character evolution. Journal of Plant Research. 116:281–294. Kimoto Y, Tobe H. 2008. Embryology of Hortonioideae and Monimioideae (Monimiaceae, Laurales): characteristics of the ‘lower’ monimioids. Botanical Journal of the Linnean Society. 158:228–241. Le-Quesne C, Stark D. 2006. Gomortega keule (Mol.) Baillon. In: Donoso C, editor. Las especies arbóreas de los bosques templados de Chile y Argentina, Autoecología. Valdivia: Marisa Cuneo Ediciones; p. 277–284. Lüttge U. 2012. A contest of lipids: the oil-carbohydrate-protein complement of plant seed storage. European Journal of Lipid Science and Technology. 114:101–102. Maldonado C. 1990. Caracterización del hábitat de Gomortega keule (Mol.) Baillon en su rango de distribución y algunos antecedentes de su reproducción sexuada [dissertation]. Universidad de Concepción. p. 126. Morales E, Calquín R. 2004. Experiencia en germinación en Queule (Gomortega keule (Mol.) Baillon). Mundo Forestal. 4:22–25. Muñoz R. 1986. El queule, una especie en peligro de extinción. Chile Forestal. 134:16–18. Muñoz-Concha D, Garrido-Werner A. 2011. Ethnobotany of Gomortega keule, an endemic and endangered Chilean tree. New Zealand Journal of Botany. 49:509–513. Myers N, Mittermeier RA, Mittermeier CG, Da Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature. 403:853–858. Orellana C. 1996. Efecto del ácido giberélico (GA3) sobre la germinación de semillas de queule (Gomortega keule (Mol.) Baillon) [dissertation]. Universidad de Talca. p. 68. Prego I, Maldonado S, Otegui M. 1998. Seed structure and localization of reserves in Chenopodium quinoa. Annals of Botany. 82:481–488. Reiche K. 1896. Zur Kenntniss von Gomortega nitida R. et Pav. Berichten der Deutschen Botanischen Gesellschaft. 14:225–233. Rodríguez G, Rodríguez RR, Barrales HL. 2007. Plantas Ornamentales de Chile. Concepción: Editora y Gráfica Lamas. p. 236. Sánchez A, Kirchner FR, Paulín N, Usami CR, López E. 1987. Cultivos oleaginosos. México: Editorial Trillas. p. 72. Schneider H, Thürmer F, Zhu JJ, Wistuba N, Gesser P, Lindner K, Herrmann B, Zimmermann G, Hartug W, Bentrup F-W, et al. 1999. Diurnal changes in xylem pressure of the hydrated resur- rection plant Myrothamnus flabellifolia: evidence for lipid bodies in conducting xylem vessels. New Phytologist. 143:471–484. Serra M, Gajardo R, Cabello A. 1986. Ficha técnica de especies amenazadas: Gomortega keule (Mol.) Baillon “Keule” (Gomortegaceae), especie en peligro. Santiago: Corporación Nacional Forestal y Universidad de Chile. p. 18. Thornley JHM, Johnson IR. 1990. Plant and crop modelling. New York: Oxford University Press. p. 669. Wang TL, Bogracheva TY, Hedley CL. 1998. Starch: as simple as A, B, C? Journal of Experimental Botany. 49:481–502.