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 sub- sequently 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 abun- dance 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-pot- assium 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.