Biochemical , histochemical and ultrastructural characterization of Centrolobium robustum ( Fabaceae ) seeds

(Biochemical, histochemical and ultrastructural characterization of Centrolobium robustum (Fabaceae) seeds). Centrolobium robustum seed coat is made up of an external layer of macrosclereids and an internal layer of osteosclereids, followed by a layer of dead cells. The endosperm is closely united to the seed coat and is made up of up to three layers of living cells rich in lipid and protein bodies. All the embryonic cells that form the different tissues of the cotyledons and embryonic axis, including the apical meristematic tissues, are storage tissues since they accumulate mainly lipids and proteins, in the form of lipid and protein bodies, water-soluble polysaccharides, and starch. In the seed coat, lignin, simple phenols and hydrolysable tannins were chemically detected.

During the mid-period of the dry season, when the availability of food becomes notoriously scarce, seeds of different species become a source of food and are widely consumed by the fauna.Justiniano & Frederickse (1998) report observations in Bolivian forests of certain vertebrates breaking open the fruits of Centrolobium microchaete to consume the seeds; these include Pyrrhura moline (parrot gris), Cebus apella (monkey) and Sciurus bolivianensis (squirrel).
The species Centrolobium robustum are tall leafy trees that can reach up to 35 m in height, occupying the dominant and co-dominant levels in the tree stratum.According to their successional status they may be classifi ed as heliophyte pioneer species or as early secondary species (Lorenzi 1992).This species is found in "cerrados" or "capoeirões", gallery forests, humid and semi-humid forests, semideciduous forests of the coast and mountain-maritime areas and in regions with medium to good soil fertility (Vidal 1978, Lorenzi 1992).
The fruit is a samara of up to 25 cm in length, surrounded by a paranuclear coriaceus wing and a seminiferous nucleus globular in form, fibrous in structure and covered with numerous spikes (Vidal 1978, Justiniano & Frederickse 1998).The fruit contains from one to three seeds separated by transversal septa and located in a single locule, forming monospermic chambers (Vidal 1978) surrounded by fi brous-woody mesocarp tissue.The seed measures from 12 to 23 mm in length and from 9 to 10 mm in width, and has the consistency of a nut.It is surrounded by a paper-like tegument, reddish-brown to dark brown in colour (Vidal 1978).The embryo is constituted by a large hypocotyl-radicle axis with a poorly differentiated plumule, and two fl eshy, planeconvex and asymmetrical cotyledons (Oliveira 1999).
The aim of the present study was to investigate Centrolobium robustum seed structure and ultrastructure and the spatial localization of food reserves.In order to produce a complete study of the seeds, we have used different but complementary methods.Along with a structural investigation using light microscopy, scanning electron microscopy and transmission electron microscopy biochemical studies for determination of lignin, phenols, tannins, protein and lipids composition were also carried out.To our knowledge, this is the fi rst study on Centrolobium robustum seed.No studies have been made of the seeds of other species of the genus, particularly with regards to the nature of their storage compounds and nutritional value.This study was done as a prelude to physiological studies on seed structure behaviour.

Materials and methods
Plant material -The fruits of C. robustum were collected on the campus of the Universidade Estadual de Campinas, SP, Brazil, between August and September of 2005, and the seeds were carefully manually removed from the fruit.Immediately after collection, seeds had 5.2% (±0.44) water content and showed 100% germination.Water content (percentage wet weight basis) of the whole seed and the isolated embryos were determined on three replications of fi ve seeds by the ISTA (2005) oven methods (103 ºC ± 1 for 17 h).In these conditions, seeds were considered mature.For histochemical and ultrastutural analysis, samples were fi xed, immediately following collection.Other seeds were kept in a freezer, at -20 ºC, until use for biochemical analysis.
Starch and hemicelluloses were stained using the periodic acid Schiff (PAS) reaction preceded by aldehyde blockage with dimedone (Sigma D 3504), and hemicelluloses identifi ed by fl uorescence after application of calcofl uor white (M2R, Polyscience) and by epifl uorescence (excitation fi lter 365-380 nm; emission fi lter: 435-475 nm) (DeMason 1986).The presence of lipids was detected by epifl uorescence (excitation fi lter: 520-560 nm; emission fi lter 510 nm) after reaction with Nile Blue A (Sigma N5632).Preparation of tissue for light microscopy (LM) and transmission electron microscopy (TEM) -Excised embryos were cut to obtain separate samples of axis, cotyledons, endosperm and seed coat and endosperm.Samples were immediately fi xed for 2 h at 4 °C, using 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and washed three times with the same buffer.Then, samples were post-fi xed in OsO 4 (1.0% in the buffer) for 2 h, dehydrated through a graded ethanolpropylene oxide series and embedded in Spurr's resin, according to Harris et al. (1994) and Kuo (2007).Semithin sections (1 μm thick) and ultrathin sections for LM and TEM, respectively, were obtained with an ultramicrotome (Reichert-Jung, Vienna, Austria) with a glass knife in buffer.Sections were mounted on grids, stained with uranyl acetate, followed by lead citrate, and examined under a TEM (JEOL 1200EX II -JEOL Ltd, Akishima, Japan).Preparation of tissue for scanning electron microscopy (SEM) -Sections of seeds cut longitudinally or transversely were fi xed as described for TEM and dehydrated through a graduated ethanol series and exchanged in isopentyl acetate.Then they were subjected to critical-point drying, followed by mounting on aluminium stubs with double-sided adhesive, sputter coated with gold and examined under a SEM (JEOL JSM 5880LV -Japan) (Coimbra & Salema 1994).Biochemical analyses -For determination of reserve compounds, the seed coat was separated from the embryo.Samples of 1.0 g of each tissue were homogenized with 25 mL of methanol-chloroformwater (MCW, 12:5:3 v/v/v) (Bieleski & Turner 1966).After centrifugation and phase separation following further addition of chloroform (1 vol.) and water (1.5 vol) to the extract supernatant (4 vol), total soluble sugars were determined in the aqueous phase by the phenol-sulphuric acid method (Handel 1968).The residue obtained after centrifugation of the initial extract was divided into two equal parts.One part was homogenized with 0.1 N NaOH to determine total soluble protein content (Bradford 1976), and the other part homogenized with 10% trichloro-acetic acid to extract water-soluble polysaccharides, followed by 30% perchloric acid for starch extraction.Both these components were determined by the anthrone method described by Graham & Smydzuk (1965).
Free lipid content was determined according to Gemmrich (1977) and lignin, after acid hydrolysis, according to Hatfield et al. (1994).Total tannin content was estimated by difference between total phenols (Singleton et al. 1999) and simple phenols.The latter determined with the Folin-Ciocalteu reagent, after precipitation of the tannins with polyvinilpolypyrrolidone (PVPP).The levels of condensed tannins were determined on aliquots of the aqueous extract obtained in the previous step, using the Stiasny reaction (Wissing 1955): (Abs (550 nm) × 78.26 × dilution factor) / (TE) Where: Abs (550 nm) = Absorbance reading at 550 nm; 78.26 = the Stiasny number; dilution factor = number of times the crude extract was diluted; TE = level of total extractives, calculated as follows: [(Initial mass -Final mass) / Final mass] × 100 The data transformed in percentage represents the level of condensed polyphenols in the extract.

Results
Seed coat -At the mature seed stage, the seed coat of C. robustum was observed to be composed of two sclereid layers, the outer, constituted by macrosclereids, and an internal layer of osteosclereids (fi gure 1A, B).The remaining tissues of the outer integument and all the cellular layers derived from the inner integument do not persist in the mature seed; only some parenchymatous layers of dead cells remain.Cellulose and hemicellulose of the cell walls were detected through metachromatic staining with PAS and by epifluorescence with Calcofl uor (excitation fi lter 365-380 nm; emission fi lter: 435-475 nm) (fi gure 1B).
SEM revealed that the surface of the C. robustum seed coat was rough, undulated, with shallow depressions (fi gure 1C).Stomata were observed on the external epidermis of the outer integument of the C. robustum seed (fi gure 1D).Endosperm -An endosperm closely linked to the seed coat was observed.Endosperm was constituted by one to two layers of living cells with relatively thick walls and the remains of dead and fl attened cells (fi gure 2A).The intense fuchsia staining of the PAS reagent detected the presence of hemicellulose in the endosperm cell walls.Histochemical tests and TEM observations revealed that the living cells of the endosperm contained a nucleus and that the cytoplasm was rich in lipid bodies and protein bodies (fi gure 2B).Embryo -All cells that make up the different tissues of the embryo of C. robustum, including the apical meristematic tissues, accumulated mainly lipids and proteins in the form of lipid and protein bodies (fi gure 2B, C).As with the endosperm, protein bodies stained blue with toluidine blue and fl uoresced with the ANS fl uorochrome (fi gure 2D).The lipid bodies fl uoresced yellow-green with the fl uorochrome Nile Blue (data not shown).On staining the cotyledon cells with Calcofluor White, their fine walls fl uoresced blue, indicating the presence of cellulose and hemicellulose (fi gure 2E).They also reacted with PAS, staining fuchsia.
Lipid bodies were observed surrounding the protein bodies.The lipid bodies of the cotyledonary cells seeds varied in size from 1.5 to 25 μm.It was observed that the protein bodies of cotyledonary cells consisted of a proteinaceous matrix enclosing globoid crystals of different sizes (fi gure 3A, B).Cytoplasm was very reduced and nuclei were relatively large and lobed (fi gure 3A-D).De-differentiated organellas, i.e. mitocondria and proplastids were diffi cult to identify because the poorly defi ned cristae in mitochondria (fi gure 3C), and absences of grana in proplastids.Biochemical analysis -Lignin was present in the seed coat, at 445.6 g kg -1 fresh mass, corresponding to 64.7% of the embryo value (fi gure 4A).Simple phenols were   found to be present in the seed coat, at 114.4 g kg -1 fresh mass, corresponding to 95.2% in relation to the embryo, together with hydrolysable tannins, at 57.1 g kg -1 fresh mass, corresponding to 86.4% of the embryo value (fi gure 4B) and condensed tannins (fi gure 4C), at 25.2%, according to the Stiasny reaction.
From the biochemical analyses made of the seed reserves of C. robustum it was found that the lipids correspond to more than 68% of the reserves, both in the integument and the cotyledons.With regard to total soluble proteins in C. robustum seeds the highest level was found in the cotyledons with 21%, while the tegument had 11%.Other reserves such as polysaccharides, total sugars and starch, the levels found were relatively small, compared with lipids and proteins (fi gure 5A, B).

Discussion
The two outer layers of the outer integument, i.e. the macrosclereid with the typical light line, and the subjacent osteosclereid layer form seed coat constituted the seed coat in the mature seeds of C. robustum.Cell walls of the remaining tissues of the outer integument and all the cellular layers derived from the inner integument persist in the mature seed.

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All these characteristics coincide with those known for seeds of legumes (Gunn 1981).
According to Beltrati & Paoli (2003), a seed can be denominated testal, when its main layer of mechanical tissue is present in the external epiderm and exotestal when the external epiderm forms a rigid palisadal layer made up of macrosclereid-type cells with thick lignifi ed walls elongated longitudinally.Considering these characteristics together with the observations here made, the seed of C. robustum could be classifi ed as belonging to the exotestal type of a testal seed.
The surface of the C. robustum seed coat was rough, undulated, with shallow depressions.According to the terms adopted by Zeng et al. (2004), this type of surface can be described as rugose-foveate.Lersten (1979) reports that seeds of the Vicieae consistently show a papillose pattern on their testae.The SEM images of C. robustum testa revealed also a papillose patterns.
Stomata were observed on the external epidermis of the outer integument of the C. robustum seed.According to Beltrati & Paoli (2003), stomata are observed on the external epidermis of the seed coat of about 30 families of the angiosperms.Normally the stomata are localized on the leaves of plants, where they regulate gas exchange with the atmosphere and function as pores, but according Rugenstein & Lersten (1981) they are rarely found in seeds.Rugenstein & Lersten (1981) fi nd stomata in the irregularly reticulate of the mature seed epidermis of eight Bauhinia species.Rosa et al. (2002) cite the presence of stomata on the epidermis of the seed coat close to the raphe of Pisonia aculeata L. (Nyctaginaceae), and Teixeira et al. (2004) mention that the presence of stomata and the absence of osteosclereids in the seed coat of the Caesalpinia echinata seed are related to a faster rate of germination.Nevertheless, little is known of the function of these stomata in seeds, but probably they allow gas exchange for photosynthesis during seed development, but before seed tissues degreening.
Endosperm -Although Vidal (1978) has classifi ed C. robustum seed as exalbuminous, we observed an endosperm closely linked to the seed coat.The greater part of the endosperm was consumed during seed development.A reduced endosperm is also found in other angiosperm species that have also been classifi ed as exendospermous, such as, species of all tribes of Asteraceae (Compositae), whose an endosperm of 1 to 3 layers envelops the embryo.Nevertheless, according to Grau & Hopf (1985), the occurrence of an endospermal tissue in this family in the taxonomical literature is widely neglected.In Bulnesia (Zygophyllaceae), whose endosperm is observed in different proportions and with different ultrastructural profiles (Maldonado et al. 1998); Chenopodium quinoa (Chenopiaceae), where the endosperm is present only in the micropylar region and consists of one or two layers of thick-walled cells surrounding the hypocotyl-radicle axis of the embryo (Prego et al. 1998); and other legume species.In Fabaceae s.l., seeds may or may not possess a discernible endosperm that varies from a trace adnate to the inner integument and adjacent to the radicle, to an endosperm that encases the embryo and is as thick as or thicker than the cotyledons.A massive endosperm (often massive amounts) is phylogenetically associated with more primitive species (Gunn 1981).Therefore, a reduced endosperm is associated with more advanced species Figure 5. Relative content (%) and concentration of reserves present in the Centrolobium robustum seed.Seed coat (A).Cotyledons (B).(Grau & Hopf 1985).On the other hand, the legume endosperm may be homogeneous or heterogeneous (Reid & Bewley 1979).In the homogeneous type, the endosperm is totally composed of living storage cells, while in the heterogeneous type only the outer layer is made up of living cells and the rest are dead cells with a lumen totally occupied by galactomanans (Reid & Bewley 1979, Reid 1985, Reid & Edwards 1995, Reid et al. 1995, Edwards et al. 2002).The endosperm of the C. robustum seed was the heterogeneous type.
Storage reserves in embryo and endosperm tissues -In C. robustum, the endosperm and embryo cells were observes fi lled with lipids and protein bodies and with nuclei irregular in form, pressured by protein and lipid bodies.In general, the mitochondrias and plastids were seen dedifferentiated.According to Vertucci & Farrant (1995) during the latter stages of storage reserves accumulation, in orthodox seeds there is a general dedifferentiation of subcellular organelles such as mitochondria, and chloroplasts.Degreening (loss of thylakoid membranes and chlorophyll) of orthodox embryos is coincident with the onset of extreme desiccation tolerance.The reduction of membrane surface area in organelles does not imply that these organelles become dysfunctional.The reduction in internal membrane surface may be, according to Vertucci & Farrant (1995), is a mechanism to slow metabolism and avoid the physical consequences of drying highly membranous system.In C. robustum embryo tissues, acquisition of desiccation tolerance (mature seeds with the very low water content, i.e. 5.2% (± 0.44)) together with subcellular feature (i.e.de-differentiation of organelles) are indicating desiccation tolerance, and anticipating an orthodox behaviour for these seeds.A detailed study of mitochondria of Phaseolus vulgaris cotyledons, made by Opik (1965), shows that mitochondria in the cotyledons are de-differentiated.The shape of the nucleus in embryo cells appears to depend on the tissue in which it occurs.In the storage cells the nuclei are usually large and lobed as in other species of Fabaceae, such as Phaseolus vulgaris (Opik 1965) and Vicia faba (Briarty et al. 1970).
As above-mentioned, endosperm and embryo cells of the C. robustum embryo accumulated lipids and proteins in the form of lipid and protein bodies and protein bodies enclosed globoid crystals.Embryos of C. robustum also stored starch, but in relatively lower quantities.
The lipid bodies of the cotyledonary cells of C. robustum seeds varied in size from 1.5 to 25 μm and are therefore within the size range reported by Murphy & Vance (1999), who observed values between 0.1 to 50 μm.The latter authors mention that the lipid bodies are dispersed in the cytoplasm or aggregated in large clumps, easily recognized as protoplasmatic inclusions.On the other hand, Huang (1992) suggests that the size of these lipid bodies depends on the species as well as environmental and nutritional factors.Vertucci & Farrant (1995) report that lipid bodies are deposited along the periphery of the plasmalemma in a number of species and suggest that they would serve as reservoirs for lipid that will be required when the plasmalemma expands during imbibition and germination.
As above-mentioned, in seeds of C. robustum, the lipid and protein bodies were found both in the cotyledons and endosperm.Moloney (1999) observes that lipid bodies in endospermous seeds of dicotyledonous plants accumulate mainly in the endosperm while Huang (1992) reports that in exendospermous seeds of dicotyledons these lipid bodies are found mainly in the cotyledons and the embryo axis.In part, these observations would be valid for C. robustum seeds, except in that lipid bodies are present in both endosperm and embryo.
The occurrence of starch grains in cotyledons depends on the species.They occur rarely in the oilstoring seeds but are common in some cotyledons, notably of legumes.In legume seeds, cotyledons store protein, lipids and starch, although the proportions vary for different species.For example, in broad bean (Vicia faba) the ratio of protein:lipid:starch is 23:1:56, but in Arachis hypogaea (peanut) it is 31:48:12 (Bewley & Black 1994).
Biochemical analysis -Lignin was present in the seed coat, and according to Taiz & Zeiger (2006) is associated with hemicellulose, providing mechanical support for this structure.On the other hand, simple phenols are found to be present in the seed coat, as well as hydrolysable tannins and condensed tannins.Condensed tannins are a group of polymeric phenols that, together with lignins, confer defence properties towards attack by bacteria, fungi, viruses and insects (Temmink et al. 1989).According to Monteiro et al. (2005), the tannins have the function of inhibiting herbivorous organisms, since at high concentrations they become unpalatable to phytophages.The authors also suggest that the tannins, in combination with some proteins, render plant tissues strongly resistant to putrefaction.Furthermore, the tannin content can vary according to the environmental, climatic, and geographic conditions, as well as to the restriction of soil nutrients or effects of atmospheric pollution (Monteiro et al. 2005).According to Laurena et al. (1984), the condensed tannins are related to the colour of the testa or seed coat.This can vary from white to yellow, red, brown and black.They also report that in the testa of Vigna unguiculata, the content of condensed tannins in the mature seeds varies from 14.7% for a reddish seed coat to 5.5% for a white seed coat.On the other hand, Guzman- Maldonado et al. (1996) report that condensed tannins in the seed coat of Phaseolus vulgaris varied from 28.5% for black to 37.4% for yellow seed coats.The high level of condensed tannins in the integument of C. robustum appears to be responsible for its typical reddish colour.
The seed reserves guarantee the survival of the seedlings during the initial stages of growth, until a capacity for photosynthesis is attained.Physically, the lipid bodies present a large surface area that permits rapid mobilization of these reserves during seedling growth (Huang 1992).Murphy et al. (2001) observe that the mobilization of neutral lipids, stored within the lipid bodies, takes place after germination.Huang (1992) mention that the majority of angiosperm seeds, especially those of legumes, store high concentrations of lipids as reserve material.According to Barclay & Earle (1974) this type of seed is also rich in proteins.The high level of lipids in the embryo of C. robustum seeds was in the range of other seeds classifi ed as oilseeds, as for example, Bertholletia excelsa (Lecythidaceae) (70% fat), Prunus dulcis (Rosaceae) (54% fat), Arachis hypogaea (Fabaceae) (50% fat), Anacardium occidentale (Anacardiaceae) (43.4% fat), Cocos nucifera (Arecaceae) (41.6% fat), Myristica fragans (Myristicaceae) (33% fat), Glycine max (Fabaceae) (17.7% fat) (http://pt.wikipedia.org;http://www.hort.purdue.edu).Otegui et al. (1998) observe a protein content in the embryo of Myrsine of 3.4%, whereas Pozuelo et al. (2001) report that the embryo of Lupinus seeds contain between 30-40% of proteins.According to Taiz & Zeiger (2006), the main function of these reserve proteins, both in the seed coat and embryo, is to supply amino acids for the formation of enzymes during germination, which are utilized by the cells to digest their own protein and lipid reserves.
According to Barclay & Earle (1974), when a seed has a high lipid and protein content, the levels of starch are low, which corresponds to that found in seeds of C. robustum, as clearly shown by the biochemical and epifl uorescent analyses.Beltrati & Paoli (2003) report that starch grains can be found in the endosperm associated with proteins in amorphous granules.Although chemical analysis of the tegument of C. robustum showed the presence of starch, this presumably was in the endosperm tissue that is closely associated with the tegument.
Microscope.We thank Dr. Ladaslav Sodek for reviewing the manuscript, and CNPq and FAPESP for fi nancial support.

Figure 1 .
Figure 1.Transverse section of Centrolobium robustum seed coat, using Calcofl uor White as fl uorochromes and observed by epifl uorescent microscopy (excitation fi lter 365-380 nm; emission fi lter: 435-475 nm).It can be seen that all cell wall were fl uorescent (A).SEM (Scanning electron microscopy) of a transverse section of the seed coat (B).SEM of the seed coat surface.It can be observed that surface is rugose-foveate, with undulations, shallow depressions and small cavities (C).SEM of external epidermis of the outer integument, showing presence of stomata (D).ct = cuticle of the seed coat; dc = dead cells, remains of the internal layers to the outer integument and of the inner integument; dec = digested endosperm cells; ec = living endosperm cells; ect = endosperm cuticle; ms = macroesclereids; oc = osteosclereids; st = stomata.

Figure 2 .
Figure 2. Micrographs of the Centrolobium robustum endosperm and inner integument.Thick cell walls of the endosperm reacted with PAS (A).Micrograph showing details of the content of the living endosperm layer and underlying collapsed layers, stained metachromatically with toluidine blue.In cells of the endosperm living layer can be seen (B).Micrograph of a cotyledonary section, stained with toluidine.Protein bodies and lipid bodies can be seen (C).Epifl uorescence of protein bodies using ANS (excitation fi lter: 395 nm; emission fi lter: 500 nm)(D).Epifl uorescence of cell walls (arrows) of the embryo using Calcofl uor White (excitation fi lter 365-380 nm; emission fi lter: 435-475 nm) (E).cw = cell wall; dec = digested endosperm cells; dic = dead cells of the inner integument; ec = living endosperm cells; ect = endosperm cuticle; emb = embryo; fec = layer of fl attened endosperm cells; lb = lipid bodies; nuc = nucleus; pb = protein bodies; sc = seed coat.

Figure 3 .
Figure 3. Transmission electron micrographs of sections of Centrolobium robustum embryo cells.Part of a cell showing lipid and protein bodies.Empty areas included in protein body matrix contained globoid crystals before they were chipped out during fi xation or sectioning (A).In detail, protein bodies, lipid bodies and areas that had been contained globoid crystals (B).Detail of a group of de-differentiated mitochondria, lipid bodies, nucleus cointained in cytoplasm (C).Detail of part of a nucleus and lipid bodies (D).c = cytoplasm; gc = globoid crystals; pb = protein bodies; m = de-differentiated mitochondria; lb = lipid bodies; nuc = nucleus.