Carbohydrate mobilisation in germinating seed of Enterolobiumcontortisiliquum and Peltophorum dubium (Fabaceae),two tropical trees used for restoration

Marina Belloni VeronesiA, Kelly SimõesA, Nelson Augusto dos Santos-JuniorB

and Marcia Regina BragaA,C

APlant Physiology and Biochemistry Department, Institute of Botany, PO Box 68041, São Paulo,04045-972, SP, Brazil.

BSeed Department, Institute of Botany, PO Box 68041, São Paulo, 04045-972, SP, Brazil.CCorresponding author. Email: bragamr@ig.com.br

Abstract. Enterolobium contortisiliquum (Vell.) Morong. and Peltophorum dubium (Spreng.) are two leguminousspecies native to Brazil that are frequently used to restore degraded areas. Seed of E. contortisiliquum areexalbuminous, whereas seed of P. dubium have a mucilaginous endosperm and both are orthodox, dormant and have awater-impermeable seed coat. There is little information about the dynamics of their germination and understandingthis process is important for propagation, conservation and satisfactory practices for restoration of degraded areas. Thus, inthis study we evaluated and compared the carbohydrate mobilisation of the seed of both species during germination andearly seedling development. Data obtained showed differences in the composition and in the mobilisation of the storagecarbohydrates in the studied species.Whereas the main storage of the E. contortisiliquum embryo is starch, the main reservefound in P. dubium is the galactomannan stored in the endosperm. The carbohydrates first hydrolysed in both species areraffinose family oligosaccharides that are used in the embryo development during germination. Starch found in cotyledonsof E. contortisiliquum or accumulated after galactomannan degradation in the embryo of P. dubium is not used duringgermination and early seedling growth in either species.

Additional keywords: galactomannan, legume, newly germinated seed, raffinose-family oligosaccharides, starch.

Received 4 October 2013, accepted 31 March 2014, published online 12 May 2014

Introduction

Carbohydrates as well as proteins and lipids are complexcompounds stored in seed that are promptly mobilised duringand after germination. They are used as building blocks and asan energy source during seedling development until theautotrophic stage is achieved (Pritchard et al. 2002). In severalorthodox seed, which are tolerant to desiccation until 10% ofthe seed water content is reached (Roberts 1973), raffinosefamily oligosaccharides (RFO) are the main non-structuralcarbohydrates used to sustain metabolic activity precedingradicle emergence (Downie and Bewley 2000; Obendorf andGórecki 2012; Sánchez-Linares et al. 2012). RFO accumulationin seed has been frequently associated with desiccation toleranceduring maturation and with seed longevity during storage(Bernal-Lugo and Leopold 1992; Leduc et al. 2012). Duringgermination, the decrease of RFO to negligible amountsindicates that they function as fast-use reserves to providecarbon to the embryo (Buckeridge et al. 2004). RFOmobilisation occurs by the action of hydrolytic enzymes, suchas invertases, which are responsible for breaking sucrose apart,

and a-galactosidase, which removes galactosyl units fromraffinose, stachyose and verbascose (Buckeridge et al. 2004;Obendorf and Górecki 2012). This process occurs directlyafter tissue hydration (Bewley and Black 1994) and, in manyspecies, these hydrolases are found in quiescent seed, indicatingthat they were synthesised during maturation and used at thebeginning of the embryo growth (Ferreira and Borghetti 2004).

In some seed, mannose-containing polysaccharides aredeposited in thickened cell walls of storage tissues andmobilised during early seedling growth (Rodríguez-Gacioet al. 2012). Galactomannans are polymers composed of amain chain of b1,4-mannosyl residues branched with a1,6-galactosyl units. They are abundant in the endosperm ofleguminous seed representing up to 30% of the total seed dryweight in many species (Buckeridge 2010; Prajapati et al.2013). Besides functioning as storage compounds, thesepolysaccharides are also important for protecting germinatingembryos from desiccation, because galactomannans are highlyhygroscopic molecules, contributing to water retention(Buckeridge 2010; Rodríguez-Gacio et al. 2012). Embryos

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play a role in galactomannan breakdown, producing a chemicalsignal for mobilisation and serving as a sink for the hydrolysisproducts, which are used as a source of carbon and energy tothe embryo metabolism (Buckeridge and Dietrich 1996; Dirket al. 1999). In fenugreek seedlings, sugars resulting fromgalactomannan degradation were taken up from the endospermby the embryo, leading to a transient starch accumulation in thecotyledons (Bewley et al. 1993). The build-up of starch in theembryo allows continued galactomannan mobilisation inthe endosperm while also avoids an increase in embryoosmotic potential (Dirk et al. 1999).

Fabaceae is one of the largest eudicotyledonous plantfamilies, containing many species that store carbohydrates asthe main reserve in their seed (e.g. Buckeridge and Dietrich1996; Garcia et al. 2006; Obendorf and Górecki 2012).Although almost 10% of leguminous species (~2740 spp.) arefound in Brazil (http://floradobrasil.jbrj.gov.br, verified 14March 2014) the mobilisation of sugars during germinationand early seedling establishment of native Brazilian species isunderstudied. Understanding the dynamics of germination ofthese species in relation to their storage compounds isimportant for propagation, conservation and use for therestoration of degraded areas. The lack of knowledge about theseed attributes that make seedlings of native species efficientand competitive in recovering deforested areas represents alimiting factor for the development of satisfactory recoverypractices (Figueiredo et al. 2012).

Enterolobium contortisiliquum (Vell.) Morong., known as‘tamboril’, is a native tree from Brazil (Aquino et al. 2009),described as a non-pioneer and frequently found colonising forestborders (Durigan et al. 2002). These seed are exalbuminous,storing carbohydrate and proteins as reserves in the cotyledons(D. A. Soares, L. R. O. Normando, M. I. Gallão, unpubl. data).Peltophorum dubium (Spreng.) Taub., known as ‘canafistula’, isalso a native arboreal species from Brazil that occurs in semi-deciduous forest (Donadio andDemattê 2000;Wanli et al. 2001).This heliophytic tree is generally found in forest clearings, beingconsidered as a pioneer species in the ecological succession(Wanli et al. 2001; Lorenzi 2008). Its seed have amucilaginous endosperm, as with most leguminous species,where carbohydrate reserves are stored (Buckeridge andDietrich 1996; Donadio and Demattê 2000). Both species areorthodox and show dormancy because of seed coatimpermeability (Wielewick et al. 2006; Guimarães et al.2011). E. contortisiliquum produces, on average, ~4600 seedkg–1, and P. dubium, whose seed are smaller, ~21 600 seed kg–1

(Davide et al. 1995). Because E. contortisiliquum and P. dubiumseed have different storage tissues, knowledge aboutmobilisation can help understand the relationship betweenstorage compounds and the germination process. Moreover,E. contortisiliquum and P. dubium are widely used inecological restoration programs (Mattei and Rosenthal 2002;Max et al. 2004; Meneguello and Mattei 2004; Santos Junioret al. 2004; Vilas Boas et al. 2004; Soares and Rodrigues2008; Costa et al. 2010; Malavasi et al. 2010; Assis et al.2011), especially in the state of São Paulo, Brazil.

The present paper reports our evaluation and comparison ofthe amount and composition of seed storage carbohydrates ofE. contortisiliquum and P. dubium during germination and early

seedling development, aimed at extending knowledge about thegerminative process of tropical legumes used for restoration ofdegraded areas.

Materials and methodsPlant materialSeed of E. contortisiliquum and P. dubium were obtained in2011–2012 from plants cultivated under natural conditions inthe gardens of the Instituto de Botânica in São Paulo, Brazil.

Seed germination and samplingSeed of E. contortisiliquum and P. dubium were scarified, oneby one, by abrading the seed coat with a grinding machine(Mechanical stirrer Fanem Contrac 1000 – emery-coupled, SãoPaulo, Brazil), followed by disinfection with an aqueoussolution of commercial sodium hypochlorite (10%) for 10minand exhaustive washing with autoclaved distilled water beforethe assays. The seed were then placed on two sheets of filterpaper moistened with 7mL of distilled water (containingfungicide DEROSAL, Bayer CropScience Brasil, Socorro,Brazil, 1.5mLL–1 v/v) in 15 transparent plastic boxes(11� 11� 3 cm) containing 10 seed each. Seed were coveredwith wet filter paper, with germination assays conducted at25� (2)�C and a photoperiod of 12 h in a germinationchamber (BOD Marconi, Piracicaba, Brazil). Germination wasdaily monitored for 5 days and radicle protrusion was used asthe germination criterion.

Three samples of seed of each species (composed of triplicatesof10 seedeach)were collecteddaily and immediatelydissected toseparate the seed coat from the embryo for E. contortisiliquumseed, and the seed coat from the endosperm and from the embryofor P. dubium seed. Because of seed hardness, endosperm ofP. dulbium could not be separated from seed coat and embryobefore 1 day of seed imbibition. Fresh weights were obtainedand seed samples were dried at 60�C for 1 week in the oven, untilconstant weight was reached (Blue M- Electric, Blue Island,Illinois, USA). Moisture content was calculated by thedifference between fresh and dried samples. The dry materialwas used for carbohydrate extraction.

Carbohydrate extraction and analysisSamples of the embryos of E. contortisiliquum and embryos orendosperm of P. dubium (from the second day on) from fiveseed in triplicates of each species were powdered using a ballmill TE-350 (Tecnal, Piracicaba, Brazil). Three-hundredmilligrams of each sample were extracted using 5mL of 80%ethanol and incubated in a water bath (Ética EquipamentosCientíficos, Vargem Grande Paulista, Brazil) at 80�C for15min, with occasional stirring. This procedure wasperformed three times, with the supernatants collected aftercentrifugation at 1000g at room temperature for 15min(Sorvall Super T21, Kendro Laboratory Products, USA) andpooled, according to Carvalho et al. (2013). Ethanol wasremoved using a rotary evaporator (r-215, Buchi, Flawil,Switzerland) and the soluble carbohydrates and reducingsugars were quantified colorimetrically in the aqueous extracts,according to Dubois et al. (1956) and Somogyi (1945),

Carbohydrate mobilisation in germinating legume seed Australian Journal of Botany 133

respectively, using glucose (Sigma Aldrich, São Paulo, Brasil)as a standard.

One millilitre of the soluble carbohydrate aqueous extractswere deionised in ion-exchange columns using Dowex resins(Sigma Aldrich, São Paulo, Brazil) in cationic 50� 8 (100–200mesh) and anionic form 1� 8 (52–100 mesh) according toMello et al. (2010). Soluble sugars were analysed by high-performance anion-exchange chromatography with pulsedamperometric detection (HPAEC–PAD) in a Dionex system(ICS-3000,Sunnyvale, CA, USA) in Carbo-Pac PA1 column.Carbohydrates were eluted isocratically with 100mM NaOHwith a flow of 0.25mLmin–1, for 35min as described byLeduc et al. (2012). The retention times were compared withSigma Aldrich standards for myo-inositol, glucose, fructose,sucrose, raffinose and stachyose. Endosperm extracts ofP. dulbium were also analysed isocratically with 16mMNaOH in a flow of 0.25mLmin–1 for 30min by HPAEC–PADfor neutral monosaccharides (galactose, glucose and mannose).The retention times were compared with commercial sugarstandards (Sigma Aldrich). All analyses were performed intriplicates.

Galactomannan extraction, hydrolysis and analysisEndosperm from five seed of P. dubium (from the second dayafter the beginning of the seed imbibition) were dried andpowdered as described above, weighed and homogenised indistilled hot water (80�C; 100mL g–1 of powder) for 8 h. Theextract was filtered in nylon and submitted to precipitationwith three volumes of 99% ethanol overnight at 4�C. Afterprecipitation, the supernatant was discarded and the precipitatewas collected by filtration in nylon and washed with pureethanol and acetone. After drying at room temperature, thegalactomannan was solubilised in distilled water at 60�C andcentrifuged to 6000g per 15min at room temperature and thesupernatantwas collected, freeze-dried andweighed (Buckeridgeand Dietrich 1990). Analyses were performed in triplicates.

Galactomannan hydrolysis was performed by adding 50mLof sulfuric acid 72% (Sigma Aldrich) into 2mg of samplesinside of a glass vial. The mixture was heated at 30�C in awater bath for 45min. After that, 850mL of distilled water wasadded to the solution and samples were transferred to sealedglass ampoules. Samples were then hydrolysed in an autoclave(Fanem, São Paulo, Brazil) at 121�C for 1 h. After hydrolysis,the pH of the solutions was neutralised using an aqueoussolution of 50% NaOH and the samples were purified usingDowex columns as described above and submitted toquantification of total sugars (Dubois et al. 1956). Neutralmonosaccharides were analysed by HPAEC–PAD in a DionexICS 3000 system, and eluted isocratically in a PA1 column with16mM NaOH with a flow of 0.25mLmin–1 for 30min. Theretention times were compared standards for fucose, rhamnose,arabinose, galactose, glucose, xylose and mannose (SigmaAldrich). All analyses were performed in triplicate.

Starch extraction and analysisTen milligrams of residue from the carbohydrate-soluble sugarextraction were enzymatically extracted as described by Amaralet al. (2007). To the residue were added 0.5mL of Bacillus

licheniformis (EC 3.3.1.1, Megazyme, Ireland) thermostablea-amylase (120UmL–1) in 10mM MOPS buffer at pH 6.5,followed by water-bath incubation at 75�C for 30min. Thisprocedure was performed twice. After that, 0.5mL ofAspergillus niger (EC 3.2.1.3 Megazyme) amyloglucosidase(30UmL–1) in 100mM sodium acetate buffer at pH 4.5 wasadded, followed by water-bath incubation at 50�C for 30min.This procedure was also performed twice. The enzymaticreaction was stopped by adding 100mL of 0.8M perchloric acid.

Starch was quantified by measuring free glucose from thepolysaccharide enzymatic breakdown. To this, 20-mL volumesof samples were incubated with 300mL of Glucose PAPLiquiform (Centerlab, São Paulo, Brazil) at 37�C for 15min.After incubation, the absorbance was read using an ELISAmicroplate reader (Bio-TEK instruments, USA) at 490 nm.Analyses were performed in triplicates.

Design and statistical analysisAll the experiments were performed in a completely randomiseddesign, with at least three replicates per treatment. Data werepreviously submitted to ANOVA, and the contrasts betweenmeans were compared by Tukey’s test at the 5% probabilitylevel (Santana and Ranal 2004). The statistical analysis wasperformed by using SISVAR 5.1 (Ferreira 2011).

Results

Germination and water imbibition

The germination of E. contortisiliquum seed started 2 days afterimbibition and, on the third day, a high germination proportionwas observed (92%, Fig. 1a). High germination was observed inP. dubium seed only from the third day (76%) and this proportionincreased up to 93% by 5 days (Fig. 1b).

The moisture content increased from 10% to ~60% duringthe first day of imbibition for both species (Fig. 1c, d), followedby a gradual increase. E. contortisiliquum embryo fresh massincreased during the first 3 days and remained relatively constantfrom the fourth day onward (Fig. 1e). P. dubium embryo freshmass slowly increased over the 5days (Fig. 1f). InP. dubium seed,a decrease in the endosperm fresh mass was observed from thethird day (Fig. 1g). P. dubium embryo dry mass increased duringgermination, whereas endosperm dry mass decreased quicklyfrom the third day (Fig. 1i, j). In contrast, variationswere detectedin E. contortisiliquum embryo dry mass, with an increase on thesecond and third days (Fig. 1h).

Oligosaccharide breakdown

No significant changes were observed in the total content ofsoluble sugars in the E. contortisiliquum embryo followinggermination (Fig. 2a). In contrast, an increase in total sugarcontent occurred in P. dubium during the imbibition phase,followed by a decrease after the third day (Fig. 2b). In theendosperm of P. dubium, high content of soluble sugars wasobserved on the fourth day (Fig. 2c). An increase of reducingsugars was observed from the third day in E. contortisiliquum(Fig. 2d) and the fourth day in P. dubium (Fig. 2e), indicatingmobilisation of storage-soluble sugars in the embryos.E. contortisiliquum seed use RFO as storage carbohydrates,which represent ~20% of the embryo dry mass. These

134 Australian Journal of Botany M. B. Veronesi et al.

oligosaccharides are hydrolysed from the second day ofimbibition, coinciding with the post-germination process, anddecreased to negligible levels on the fourth day (Table 1). RFOwere also found both in the embryo (6%) and endosperm (4%of dry mass) of P. dubium seed, but in lower amounts thanin E. contortisiliquum (Table 1). Monosaccharides, mainly

represented by mannose and galactose, were also detected inthe endosperm extracts of P. dubium (data not shown).

Galactomanann breakdown and starch

The endosperm of P. dubium seed comprised mainlygalactomannan, which represents ~80% of its dry mass. After

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Fig. 1. (a, b) Germination), (c, d) moisture content, (e–g) fresh and (h–j) dry masses of embryos of Enterolobium contortisiliquum, and embryo andendosperm of Peltophorum dubium seed. Because of their hardness, P. dubium seed were not dissected before 1 day of imbibition, and endosperm were notanalysed at this period. Data represent the average n= 3 (10 seed per replicate), and bars represent the standard deviation. Values followed by the same lettersare not significantly different by Tukey’s test at P= 0.05.

Carbohydrate mobilisation in germinating legume seed Australian Journal of Botany 135

5 days from imbibitions, about half of the galactomannan hadbeen mobilised (Fig. 2f) and the content of soluble sugars hadincreased as a result of hydrolysis (Fig. 2c). Larger amounts ofsucrose, raffinose and stachyose were found at the fourth day andwere mobilised from then on (Table 1). The mannose : galactoseratio of the galactomannan in the endosperm of P. dubiumremained constant from the second to the fourth day ofimbibition (~1.5 : 1), but by the fifth day, this ratio hadchanged to 2 : 1. This change in the ratio is consistent with thedecrease in the levels of galactose during reserve mobilisation(Table 2). Besides RFO, E. contortisiliquum seed also had highstarch levels, representing ~40% of the embryo dry mass(Fig. 2g). The mobilisation of this polysaccharide was detectedafter 5 days of imbibition, suggesting its use in later stages ofseedling development. Starch was not detected in P. dubiumseed as storage carbohydrate when quiescent, but a consistent

increase of this polysaccharide was observed after radicleprotrusion (Fig. 2h).

Discussion

The leguminous species E. contortisiliquum and P. dubiumhave orthodox seed with dormancy resulting from coatimpermeability (Wielewick et al. 2006; Guimarães et al.2011). When scarified, seed of both species showed a quickincrease in moisture content and germinated from thesecond day following imbibition, reaching values close to 90%on the fifth day (Fig. 1). However, the embryo fresh-massincrease was slower in P. dubium than in E. contortisiliquum,probably owing to the control of water imbibition bygalactomannan stored in the endosperm of P. dubium.Galactomannans are considered to be bifunctional molecules,

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Fig. 2. Content of carbohydrates in the embryo of Enterolobium contortisiliquum, and embryo and endosperm of Peltophorum dubium seed. (a–c) Solublesugars, (d, e) reducing sugars, (f) galactomannan content in P. dubium, and (g, h) content of starch in both seed. Black bars in the columns graphs (g, h) representthe content found in the whole seed. Data represent the average of n= 3 (five seed per replicate). Bars represent the standard deviation. Values followed by thesame letters in columns are not significantly different by Tukey’s test at P= 0.05.

136 Australian Journal of Botany M. B. Veronesi et al.

playing a role in the control of water imbibition duringgermination and also serving as a carbon source fordeveloping seedlings (Buckeridge 2010). The presence ofgalactomannans in legume seed can contribute to improvingthe ecophysiological performance of seedlings in their naturalenvironment (Tonini et al. 2006; Ferreira et al. 2009).Considering that P. dubium is described as a pioneer speciesin the ecological succession (Wanli et al. 2001), the presence ofa galactomannan-rich endosperm may confer an advantage inenvironments with frequent changes in the water availability.This reserve represents an important seed attribute avoidingembryo desiccation in the case of seed dehydration afterimbibition.

The type and amount of seed reserves available for embryogrowth vary among species. Mature dry legume seed maycontain many different soluble sugars that are accumulatedduring physiological maturation and play a protective roleagainst desiccation. Sucrose and the RFO, mainly representedby raffinose, stachyose and verbascose, are usually the mostabundant carbohydrates in quiescent seed, and are quicklydegraded and used for radicle protusion and early seedlingdevelopment (Bernal-Lugo and Leopold 1992; Buckeridge andDietrich 1996; Garcia et al. 2006; Obendorf and Górecki 2012).Embryos of E. contortisiliquum had high amounts of RFO asstorage carbohydrates, which were hydrolysed in the beginningof imbibition and used during embryo development. Incontrast, embryos of P. dubium had low levels of RFO,which were also mobilised during germination and used in theembryo development (Table 1). As previously mentioned,E. contortisiliquum and P. dubium have both orthodox seed(Wielewick et al. 2006; Guimarães et al. 2011) and thepresence of RFO in orthodox seed has been correlated withtheir desiccation tolerance (Black et al. 1999; Leduc et al. 2012).

RFO have also been involved in seed storability. Mello et al.(2011) showed that the increase in these oligosaccharides intropical legume seed seems to be crucial for the storagebehaviour of these seed. Reports indicate that differences inseed viability between E. contortisiliquum and P. dubiumoccur when they are stored at room temperature. A decrease of~36% in seed germination was observed in P. dubium after5 months, whereas E. contortisiliquum maintained seedviability when stored up to 12 months under the sameconditions (Perez et al. 1999; Scalon et al. 2005). Therefore,the differences found in the level of RFO betweenE. contortisiliquum and P. dubium may be related, at least inpart, to the differences observed in maintaining seed viabilityduring storage.

Sucrose and stachyose contents were higher in quiescentseed of E. contortisiliquum than in P. dubium where raffinosewas the predominant RFO in embryos (Table 1). In seed ofboth species, sucrose increased following the decrease in RFO.a-galactosidases are enzymes ubiquitous in legume seedresponsible for RFO degradation and are accumulated duringseed development or synthesised after germination (reviewedby Obendorf and Górecki 2012). Although no enzymaticmeasurements were undertaken in the present research, therapid increase in the sucrose content, followed by the decreasein raffinose and stachyose from the first day in both speciesindicated that these enzymes are active from the beginning ofimbibition. Sucrose, besides being used as a storage compound,

Table 1. Content of soluble carbohydrate (mg g–1 DW) estimatedby high-performance anion-exchange chromatography with pulsedamperometric detection (HPAEC–PAD) in Enterolobium contortisiliquum

and Peltophorum dubium seed–, not analysed because of seed hardness, endosperm of P. dulbium couldnot be separated from seed coat and embryo before 24 h of seed imbibitions.Data represents the average of n= 3 (five seed per each replicate)� s.d.Values in a column followed by the same letter are not significantly

different by Tukey’s test at P= 0.05

Time (day) Sugar content (mg g–1 DW)E. contortisiliquum P. dubium

Embryo Embryo Endosperm

Sucrose0 11.2 ± (3.88)a 1.3 ± (0.34)a –

1 26.3 ± (11.06)ab 9.1 ± (1.06)ab –

2 25.6 ± (4.11)ab 12.9 ± (1.62)bc 3.2 ± (0.44)a3 47.2 ± (10.03)c 23.5 ± (1.27)d 5.5 ± (0.40)ab4 32.6 ± (6.87)bc 19.4 ± (1.73)d 17.6 ± (5.36)b5 32.2 ± (0.89)bc 14.7 ± (2.62)c 18.9 ± (9.20)b

Raffinose0 27.9 ± (8.93)ab 10.9 ± (0.65)ab –

1 77.2 ± (21.09)c 24.5 ± (5.00)cd –

2 45.4 ± (9.21)b 29.3 ± (5.48)d 1.2 ± (0.26)a3 6.2 ± (2.07)a 14.7 ± (7.47)bc 0.3 ± (0.10)a4 3.1 ± (3.15)a 2.34 ± (0.88)a 12.4 ± (7.95)a5 1.4 ± (0.42)a 0.7 ± (0.47)a 8.6 ± (9.34)a

Stachyose0 19.7 ± (5.82)a 1.0 ± (0.12)ab –

1 61.8 ± (14.79)c 3.2 ± (0.70)c –

2 40.5 ± (2.90)b 3.6 ± (0.38)c 0.5 ± (0.13)a3 15.3 ± (5.28)a 1.8 ± (0.67)b 0.3 ± (0.16)a4 4.4 ± (4.84)a 0.6 ± (0.16)ab 4.0 ± (0.64)b5 2.3 ± (1.21)a 0.2 ± (0.20)a 1.4 ± (0.77)a

Table 2. Content of monosaccharides (mg g–1 DW) estimated by high-performance anion-exchange chromatography with pulsed amperometricdetection (HPAEC–PAD) and mannose : galactose ratio in Peltophorium dubium endosperm

Because of seed hardness, endosperm of P. dulbium could not be separated from seed coat and embryo before 24 h of seed imbibition. Data represents the meanof n= 3 (five seed per each replicate)� s.d. Values in a column followed by the same letter are not significantly different by Tukey’s test at P= 0.05

Time (day) Fucose Ramnose Arabinose Galactose Glucose Xylose and mannose Mannose : galactose ratio

2 1.3 ± (0.4) a 2.8 ± (0.8)a 2.1 ± (0.3)a 33.7 ± (1.7)c 1.9 ± (0.6)a 58.2 ± (1.1)b 1.73 : 13 1.0 ± (0.2)a 1.8 ± (0.2)a 2.3 ± (0.4)a 35.6 ± (0.2)c 1.9 ± (0.4)a 57.4 ± (0.7)b 1.61 : 14 1.9 ± (1.0)ab 3.4 ± (2.6)a 11.9 ± (4.7)b 25.9 ± (4.0)b 10.0 ± (5.9)b 47.0 ± (6.0)a 1.81 : 15 2.9 ± (0.6)b 1.2 ± (0.1)a 15.4 ± (1.6)b 19.1 ± (1.2)a 18.8 ± (0.3)c 42.6 ± (0.8)a 2.23 : 1

Carbohydrate mobilisation in germinating legume seed Australian Journal of Botany 137

also plays a role in protecting plasma membrane duringdesiccation and as a signal for regulating gene expression andenzyme activities involving further carbohydrate metabolism(Bernal-Lugo and Leopold 1992; Dirk et al. 1999). Althoughraffinose is present in all mature dry legume seed as sucrose,this trisaccharide is not usually the most predominant RFO(Obendorf and Górecki 2012), as was observed here inE. contortisiliquum seed. Raffinose is known to enhance theprotective effect of sucrose during seed desiccation (Bernal-Lugo and Leopold 1992) and the high content found inE. contortisiliquum may help maintain higher seed viabilityduring storage when compared with P. dubium (Perez et al.1999; Scalon et al. 2005).

Starch is another carbohydrate commonly stored inlegume seed. Juliano and Varner (1969) found that 45% ofthe Pisum sativum L. cotyledons dry weight corresponds tothis polysaccharide. More recently, Garcia et al. (2006) found30–40%starch inCaesalpinea echinata seed.E. contortisiliquumhad starch as the main storage compound, which was equivalentto 40% of the embryo dry weight (Fig. 2). Mobilisation of thispolysaccharidewas seen from thefifth day, suggesting it is a post-germinative reserve. However, using histochemical analyses,D. A. Soares, L. R. O. Normando, M. I. Gallão (unpubl. data)found an increase in starch grains 12 h after imbibition, followedby a decrease up to 60 h in E. contortisiliquum seed. A gradualincrease of this polysaccharide was also noted in the cytoplasmthroughout the 60 h of imbibition, suggesting that it is storedfirst probably as result of RFO hydrolysis and later degradedto supply carbon to developing embryos (D. A. Soares,L. R. O. Normando, M. I. Gallão unpubl. data). Discrepanciesfound in the starchmetabolism inE. contortisiliquum in thisworkcompared with the results of D. A. Soares, L. R. O. Normando,M. I. Gallão (unpubl. data) may be related to differences inanalytical methods used between the two studies. Thecolorimetric assay used here to quantify starch does not allowthe small changes in starch content observed previously by thehistochemical analysis.

Seed that have hemicelluloses (xyloglucans andgalactomannans) as storage carbohydrates normally do notaccumulate starch in their tissues (Bewley and Black 1994).With a galactomannan-rich endosperm, quiescent seed ofP. dubium do not have starch as storage compound. However,after germination, there was an increase in the level of thispolysaccharide, probably as a result of the mobilisation of theendosperm, which provides high amounts of monosaccharidestransported from the endosperm and stored in the embryo(Fig. 2). The same pattern was found in Sesbania marginata(synonymous to S. virgata) and Trigonella foenum-graecumseed (Buckeridge and Dietrich 1996; Dirk et al. 1999).According to Buckeridge (2010), galactomannan degradationseems to occur in a non-stop mode, which explains embryostarch accumulation when carbon delivery from the endospermexceeds the embryo demand for respiration and growth.

Besides the RFO, galactomannans present in the endospermof many leguminous species are also completely hydrolysedinto galactose and mannose during germination, and absorbedand utilised as an energy source by the embryo (Buckeridgeand Dietrich 1996; Buckeridge 2010). P. dubium seed havelarge amounts of galactomannans in their endosperms, which

are hydrolysed from the third day of imbibition (Fig. 2). RFO inP. dubium endosperm were detected in large amounts only onthe fourth day following imbibition (Table 1), suggesting thatthese are synthesised following the galactomannan mobilisationduring the first days after germination. This is in agreementwith the high amounts of free mannose and galactose detectedby this analysis of the soluble sugars in the tissue (data not shown).

The mannose : galactose ratio in leguminous seed variesamong species, from a totally substituted polymer (1 : 1) inTrigonella foenum-graecum to 4 : 1 in Ceratonia siliqua(Buckeridge 2010). The 1.5 : 1 mannose : galactose ratio foundin the P. dubium endosperm was similar to that reported byBuckeridge (2010) in endosperms of seed in the genusPeltophorum and by Buckeridge and Dietrich (1996) in theendosperms of S. marginata (synonymous to S. virgata). InP. dubium, this ratio varied during germination, reachingvalues close to 2 : 1 on the fifth day, indicating differentialaction of enzymes such as a-galactosidase, b-mannanase andb-mannosidase on the mobilisation of this polymer (Table 2).

In conclusion, the data presented here showed differences inthe composition and mobilisation of the storage carbohydratesin the species evaluated that may be related to their ecology.E. contortisiliquum does not have endosperm and carbohydratesare mainly stored as starch. The mobilisation of this compoundstarts only after the hydrolysis of the RFO, 4 days afterimbibition. P. dubium, however, has a galactomannan-richendosperm as the main storage tissue. Embryos contain RFOthat are also the first carbohydrates mobilised at the beginningof imbibition. Galactomannan mobilisation was observed fromthe third day after imbibiton and provided products for starchaccumulation in P. dubium embryos, which were later usedduring seedling development. According to Buckeridge(2010), galactomannans as seed storage compounds, incontrast to starch, offers additional advantages beyondproviding carbon and energy to the growing embryo. Besidesthe importance of galactomannan in regulating the waterentrance, owing to its high viscosity and solubility, thispolysaccharide is proposed to also play a role in protectingembryos against pathogens by forming a highly viscousphysical barrier (Reid and Bewley 1979; Potomati andBuckeridge 2002; Buckeridge 2010). Moreover, in somelegume seed such as S. virgata, enzymes for galactomannandegradation are believed to be present in protein bodies, whichare synchronically degraded with galactomannan (Tonini et al.2010). Therefore, reserve mobilisation in this species would bea lower-energy process for seed because the investment toproduce the degrading enzymes would come from the motherplant (Buckeridge 2010). Considering that, it is reasonable tosuppose that P. dubium seed may have a better physiologicalperformance as a pioneer species than does other pioneervegetation bearing starch-containing seed. E. contortisiliquum,in turn, is described as a non-pioneer species frequently foundcolonising forest borders (Durigan et al. 2002) and its seedpresent starch as a main reserve, which seems to be morecompatible with this environment.

Understanding the biology of the species, in particular theknowledge of the mechanism and regulation of germination,including the participation of the reserves in the process, isimportant for supporting actions to restore degraded areas

138 Australian Journal of Botany M. B. Veronesi et al.

(Davide and Silva 2008). Our findings report storagecarbohydrates in seed from two tropical legumes and haveextended our knowledge about their germination and initialseedling-establishment requirements. This may also helpunderstand important physiological aspects for their storageand use for restoration of degraded areas. Although seed ofP. dulbium seem to offer some advantages in providingprotection and carbon to the growing embryo, their storabilityunder natural conditions is reduced. Practically, this knowledgecan lead to a selection of native plant species more appropriateand competitive for recovery of degraded areas on the basis ofthe seed storage compounds, consequently resulting in a betterseedling performance until they reach autotrophy. Additionally,it may help understand why some species are better competitorsand show invasive behaviour in revegetated areas. Morestudies that include storage and mobilisation of seed reservesin tropical native species are still necessary before a selectednumber of plant species are recommended for the developmentof satisfactory restoration practices.

Acknowledgements

This work is part of the MSc Thesis of M. B. Veronesi at Instituto deBotânica, SP (Brazil) and was supported by Fundacão de Amparo àPesquisa do Estado de São Paulo (FAPESP, 2010/14299-0) for thescholarship. Thanks are due to Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq, Grant 474325/2009-1) and Fundacão deAmparo a Pesquisa do Estado de São Paulo (FAPESP, Grants 2005/04139-7and 2012/16332-0), which supported the work. M. R. Braga acknowledgesCNPq for the research fellowship.

References

Amaral LIV, Gaspar M, Costa PMF, Aidar MPM, Buckeridge MS (2007)Novo método enzimático rápido e sensível de extracão e dosagem deamido em materiais vegetais. Hoehnea 34, 425–431.doi:http://dx.doi.org/10.1590/S2236-89062007000400001

Aquino AFMAG, Ribeiro MCC, Paula YMC, Bnedito CP (2009) Superacãode dormência em sementes de orelha-de-negro (Enterolobiumcontortisiliquum (Vell.) Morang.). Revista Verde de Agrologia eDesenvolvimento Sustentável 4, 69–75.

Assis IR, Dias LE, Abrahão WAP, Ribeiro Junior ES, Mello JWV (2011)Cover layers to the growth of trees and shrubs over a sulfide spoil fromgold mining. Revista Árvore 35, 941–947.doi:10.1590/S0100-67622011000500019

Bernal-Lugo I, Leopold CA (1992) Changes in soluble carbohydratesduring seed storage. Plant Physiology 98, 1207–1210.doi:10.1104/pp.98.3.1207

Bewley JD, Black M (1994) ‘Seeds: physiology of development andgermination.’ 2nd edn. (Plenum Press: New York)

Bewley JD, Leung DWM, Maclsaac S, Reid JSG, Xu N (1993) Transientstarch accumulation in the cotyledons of fenugreek seeds duringgalactomannan mobilization from the endosperm. Plant Physiologyand Biochemistry 31, 483–490.

Black CM, Corbineau F, Gee H, Come D (1999) Water content, raffinoseand dehydrins in the induction of desiccation tolerance in immaturewheat embryos. Plant Physiology 120, 463–472.doi:10.1104/pp.120.2.463

Buckeridge MS (2010) Seed cell wall storage polysaccharides: models tounderstand cell wall biosynthesis and degradation. Plant Physiology 154,1017–1023. doi:10.1104/pp.110.158642

Buckeridge MS, Dietrich SMC (1990) Galactomannan from Brazilianlegume seeds. Revista Brasileira de Botânica 13, 109–112.

Buckeridge MS, Dietrich SMC (1996) Mobilisation of the raffinose familyoligosaccharides and galactomannan in germinating seeds of Sesbaniamarginata Benth. (Leguminosae–Faboideae). Plant Science 117, 33–43.doi:10.1016/0168-9452(96)04410-X

Buckeridge MS, Aidar MPM, Santos HP, Tiné MAS (2004) Acúmulo dereservas. In ‘Germinacão: do básico ao aplicado’. (Eds AG Ferreira, FBorghetti) pp. 31–50. (Artmed: Porto Alegre, Brazil)

Carvalho CP, Hayashi AH, Braga MR, Nievola CC (2013) Biochemical andanatomical responses related to the in vitro survival of the tropicalbromeliad Nidularium minutum to low temperatures. Plant Physiologyand Biochemistry 71, 144–154. doi:10.1016/j.plaphy.2013.07.005

Costa MP, Nappo ME, Cacador FRD, Barros HHD (2010) Avaliacão doprocesso de reabilitacão de um trecho de floresta ciliar na Bacia do RioItapemirim-ES. Revista Árvore 34, 835–841.doi:10.1590/S0100-67622010000500009

Davide AC, Silva EAA (Eds) (2008) ‘Producão de sementes e mudas deespécies florestais.’ (Universidade Federal de Lavras: Lavras, Brazil)

Davide AC, Faria JMR, Botelho SA (1995) ‘Propagacão de espéciesflorestais.’ (Companhia Energética de Minas Gerais: Belo Horizonte,Brazil)

Dirk LMA, van der Krol AR, Vreugdenhil D, Hilhorst HWM, Bewley JD(1999) Galactomannan, soluble sugar, and starch mobilizationfollowing germination of fenugreek (Trigonella foenum-graecum L.)seeds. Plant Physiology Biochemistry 37, 41–50.doi:10.1016/S0981-9428(99)80065-5

Donadio NMS, Demattê MESP (2000) Morfologia de frutos, sementese plântulas de canafístula (Peltophorum dubium (Spreng.) Taub)e jacarandá-da-Bahia (Dalbergia nigra (Vell.) Fr.All. ex Benth) –

Fabaceae. Revista Brasileira de Sementes 22, 64–73.Downie B, Bewley D (2000) Soluble sugar content of white spruce

(Picea glauca) seeds during and after germination. PhysiologiaPlantarum 110, 1–12. doi:10.1034/j.1399-3054.2000.110101.x

DuboisM, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetricmethod for determination of sugars and related substances. AnalyticalChemistry 28, 350–356. doi:10.1021/ac60111a017

Durigan G, Nishikawa DLL, Rocha E, Silveira ER, Pulitano FM, RegaladoLB, Carvalhaes MA, Paranaguá PA, Ramilu VEL (2002) Caracterizacãode dois estados da vegetacão de uma área de cerrado no município deBrotas-SP, Brasil. Acta Botânica Brasílica 16, 251–262.doi:10.1590/S0102-33062002000300002

Ferreira DF (2011) Sisvar: a computer statistical analysis system. Ciência eAgrotecnologia 35, 1039–1042.doi:10.1590/S1413-70542011000600001

Ferreira AG, Borghetti F (Eds) (2004) ‘Germinacão: do básico ao aplicado.’(Artmed: Porto Alegre, Brazil)

Ferreira CS, Piedade MTF, Tiné MAS, Rossato DR, Parolin P, BuckeridgeMS (2009) The role of carbohydrates in seed germination and seedlingestablishment of Himatanthus sucuuba, an Amazonian tree withpopulations adapted to flooded and non-flooded conditions. Annals ofBotany 104, 1111–1119. doi:10.1093/aob/mcp212

Figueiredo MA, Baêta HE, Kozovits AR (2012) Germination of nativegrasses with potential application in the recovery of degraded areas inQuadrilátero Ferrífero, Brazil. Biota Neotropica 12, 118–123.doi:10.1590/S1676-06032012000300013

Garcia IS, Souza A, Barbedo CJ, Dietrich SMC, Figueiredo-Ribeiro RCL(2006) Changes in soluble carbohydrates during storage of seeds ofCaesalpina echinata Lam. (Brazilwood), an endangered leguminoustree from Brazilian Atlantic Forest. Brazilian Journal of Biology 66,739–745. doi:10.1590/S1519-69842006000400018

Guimarães CC, Faria JMR, Oliveira JM, Silva EAA (2011) Avaliacãoda perda da tolerância à dessecacão e da quantidade de DNA nuclearem sementes de Peltophorum dubium (Spreng.) Taubert durante e após agerminacão. Revista Brasileira de Sementes 33, 207–215.doi:dx.doi.org/10.1590/S0101-31222011000200002

Carbohydrate mobilisation in germinating legume seed Australian Journal of Botany 139

Juliano BO, Varner JE (1969) Enzymic degradation of starch granules inthe cotyledons of germinating peas. Plant Physiology 44, 886–892.doi:10.1104/pp.44.6.886

Leduc SNM, Silva JPN, Gaspar M, Barbedo CJ, Figueiredo-Ribeiro RCL(2012) Non-structural carbohydrates of immature seeds of Caesalpiniaechinata (Leguminosae) are involved in the induction of desiccationtolerance.Australian Journal ofBotany60, 42–48. doi:10.1071/BT11236

Lorenzi H (2008) ‘Árvores brasileiras: manual de identificacão e cultivo deplantas arbóreas nativas do Brasil.’ (Editora Plantarum: Nova Odessa,Brazil)

Malavasi CM, Klein J, Malavasi MM (2010) Efeito de um protetor físico nasemeadura direta de duas espécies florestais em área de domínio ciliar.Revista Árvore 34, 781–787. doi:10.1590/S0100-67622010000500003

Mattei VL, Rosenthal MD (2002) Semeadura direta de canafístula(Peltophorum dubium (Spreng.) Taub.) na regeneracão de capoeiras.Revista Árvore 26, 649–654. doi:10.1590/S0100-67622002000600001

Max JCM, Melo ACG, Faria HH (2004) Comportamento de seis espéciesnativas de dois grupos ecológicos plantadas em diferentes espacamentosem reflorestamento ciliar (cap 23). In ‘Pesquisa em conservacão erecuperacão ambiental no oeste paulista’. (Eds O Vilas Boas, GDurigan) pp. 385–395. (Instituto Florestal/Secretaria do MeioAmbiente: São Paulo, Brazil)

Mello JIO, Barbedo CJ, Salatino A, Figueiredo-Ribeiro RCL (2010) Reservecarbohydrates and lipids from the seeds of four tropical tree specieswith different sensitivity to desiccation. Brazilian Archives of Biologyand Technology 53, 889–899. doi:10.1590/S1516-89132010000400019

Mello JIO, Centeno DC, Barbedo CJ, Figueiredo-Ribeiro RCL (2011)Changes in carbohydrate composition in seeds of three tropical treespecies stored at freezing temperature. Seed Science and Technology39, 465–480.

Meneguello GE, Mattei VL (2004) Semeadura direta de timbaúva(Enterolobium contortisiliquum), canafístula (Peltophorum dubium) ecedro (Cedrela fissilis) em campos abandonados. Ciência florestal 14,21–27.

Obendorf RL, Górecki RJ (2012) Soluble carbohydrates in legume seeds.Seed Science Research 22, 219–242. doi:10.1017/S0960258512000104

Perez SCJG, Fanti SC, Casali CA (1999) Influência do armazenamento,substrato, envelhecimento precoce e profundidade de semeadura nagerminacão de canafístula. Bragantia 58, 57–68.doi:10.1590/S0006-87051999000100008

Potomati A, Buckeridge MS (2002) Effect of abcisic acid on themobilization of galactomannan and embryo development of Sesbaniavirgata (Cav). Pers (Leguminosae – Faboideae). Revista Brasileira deBotânica 25, 303–310. doi:10.1590/S0100-84042002000300006

Prajapati VD, Jani GK, Moradiya NG, Randeria NP, Nagar BJ, Nikhil NN,Variya BC (2013) Galactomannan: a versatile biodegradable seedpolysaccharide. International Journal of Biological Macromolecules60, 83–92. doi:10.1016/j.ijbiomac.2013.05.017

Pritchard SL, Charlton WL, Baker A, Graham IA (2002) Germinationand storage reserve mobilization are regulated independently inArabidopsis. The Plant Journal 31, 639–647.doi:10.1046/j.1365-313X.2002.01376.x

Reid GJS, Bewley D (1979) A dual role for the endosperm and itsgalactomannan reserves in the germinative physiology of Fenugreek(Trigoneila foenum-graecum L.), an endospermic leguminous seed.Planta 147, 145–150. doi:10.1007/BF00389515

Roberts EH (1973) Predicting the storage life of seeds. Seed Science andTechnology 1, 499–514.

Rodríguez-GacioMC, Iglesias-Fernández R, Carbonero P,Matilla AJ (2012)Softening-up mannan-rich cell walls. Journal of Experimental Botany63, 3976–3988. doi:10.1093/jxb/ers096

Sánchez-Linares L,Gavilanes-RuízM,Díaz-PontonesD,Guzmán-ChávezF,Calzada-Alejo V, Zurita-Villegas V, Luna-Loaiza V,Moreno-SánchezR,Bernal-Lugo I, Sánchez-Nieto S (2012) Early mobilization and radicleprotrusion in maize germination. Journal of Experimental Botany 63,4513–4526. doi:10.1093/jxb/ers130

Santana DG, Ranal MA (2004) ‘Análise da germinacão: um enfoqueestatístico.’ (Universidade de Brasília: Brasília)

Santos Junior NA, Botelho SA, Davide AC (2004) Estudo da germinacão esobrevivência de espécies arbóreas em sistema de semeaduradireta,visando à recomposicão de mata ciliar. Cerne 10, 103–117.

Scalon SPQ, Mussury RM, Wathier F, Gomes AA, Silva KA, Pierezan L,Scalon Filho H (2005) Armazenamento, germinacão de sementes ecrescimento inicial de mudas de Enterolobium contortisiliquum (Vell.)Morong. Acta Scientiarum Biological Sciences 27, 107–112.doi:10.4025/actascibiolsci.v27i2.1318

Soares PG, Rodrigues RR (2008) Semeadura direta de leguminosasflorestais: efeito da inoculacão com rizóbio na emergência de plântulase crescimento inicial no campo. Scientia Forestalis 36, 115–121.

Somogyi M (1945) A new reagent for the determination of sugars. TheJournal of Biological Chemistry 160, 61–68.

Tonini PP, Lisboa CGS, Silva CO, Mazzoni-Viveiros SC, Buckeridge MS(2006) Testa is involved in the control of storage mobilization in seedsof Sesbania virgata (Cav.) Pers., a tropical legume tree from of theAtlantic Forest. Trees 21, 13–21. doi:10.1007/s00468-006-0091-1

Tonini PP, Carrara TB, Buckeridge MS (2010) Storage proteins and cellwall mobilisation in seeds of Sesbania virgata (Cav.) Pers.(Leguminosae). Trees 24, 675–684. doi:10.1007/s00468-010-0437-6

Vilas Boas O, Max JCM, Nakata H (2004) Recuperacão da coberturavegetal – Crescimento e sobrevivência das mudas de essências nativasproduzidas em diferentes recipientes (cap 16). In ‘Pesquisa emconservacão e recuperacão ambiental no oeste paulista’. (Eds O VilasBoas, G Durigan) pp. 293–304. (Instituto Florestal/Secretaria do MeioAmbiente: São Paulo, Brazil)

Wanli Z, Leihong L, Perez SCJGA (2001) Pré-condicionamento e seusefeitos em sementes de canafístula (Peltophorum dubium (Spreng.)Taub.). Revista Brasileira de Sementes 23, 146–153.

Wielewick AP, Leonhoardt C, Schlindwein G, Medeiros ACS (2006)Proposta de padrões de germinacão e teor de água para sementes dealgumas espécies florestais presentes na resgião sul do Brasil. RevistaBrasileira de Sementes 28, 191–197.doi:10.1590/S0101-31222006000300027

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