Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

Reserve mobilization and secondary metabolites during seed germination and

seedling establishment of the tree Erythrina velutina (Fabaceae)

Diego Augusto Azevedo-da-Silva1; https://orcid.org/0000-0002-3280-1342

Danilo Flademir Alves-de-Oliveira1; https://orcid.org/0000-0001-8583-4132

Herley Carlos Bezerra-de-Oliveira1; https://orcid.org/0000-0002-6786-1740

Thadeu Martins Feitosa1; https://orcid.org/0000-0002-7634-9183

Ivanice Bezerra da Silva2; https://orcid.org/0000-0003-1575-5310

Raquel Brandt Giordani2; https://orcid.org/0000-0001-6500-0755

Eduardo Luiz Voigt1*; https://orcid.org/0000-0003-0481-1129

1. Departamento de Biologia Celular e Genética, Universidade Federal do Rio Grande do Norte, Natal, Brazil;

diego_augusto16@hotmail.com, flademir.oliveira@hotmail.com, herleycarl@gmail.com, tha_deu@hotmail.com,

eduardo.voigt@ufrn.br (*Correspondence)

2. Departamento de Farmácia, Universidade Federal do Rio Grande do Norte, Natal, Brazil; ivanicebsilva@gmail.com,

raquebg@hotmail.com

Received 25-V-2022. Corrected 27-IV-2023. Accepted 04-VIII-2023.

ABSTRACT

Introduction: The lack of knowledge on seed germination and seedling establishment is a main constraint for

the restoration of degraded areas, including the tropical dry forest known as Caatinga.

Objective: To assess reserve and secondary metabolite mobilization during seed germination and seedling estab-

lishment in Erythina velutina.

Methods: We scarified, disinfected, imbibed, sown between towel paper, and incubated seeds under controlled

conditions. We hydroponically cultivated seedlings in a greenhouse. We harvested cotyledons at seed imbibition,

radicle protrusion, hypocotyl emergence, apical hook formation and expansion of cordiform leaves, first trifoliate

leaf, and second trifoliate leaf.

Results: Seeds contained approximately 20 % starch, 14.5 % storage proteins, 11.6 % neutral lipids, and 5.7 %

non-reducing sugars on a dry weight basis. Soluble sugars were mainly consumed from hypocotyl emergence

to apical hook formation, while major reserves were mobilized from apical hook formation to expansion of

first trifoliate leaf. Enzymatic activity increased from mid to late seedling establishment, causing the mobiliza-

tion of starch, oils, and proteins. Terpenoid-derivatives, flavonoids, phenolic acids, and alkaloids were detected.

Flavonoids and phenolic acids were present at almost all stages and terpenoid-derivatives disappeared at expan-

sion of cordiform leaves.

Conclusion: Soluble sugars support early seedling growth, while starch, oils and proteins are simultaneously mobi-

lized from mid to late establishment by amylases, lipases, and acid proteases. The cotyledons contain secondary

metabolites, which may act in seedling defense. High content of reserves and presence of secondary metabolites in the

cotyledons could enable E. velutina seedlings endure stress, validating their use in the restoration of degraded areas.

Key words: Caatinga; heterotrophy-autotrophy transition; reserve-degrading enzymes; specialized metabolites;

storage compounds; tree legume.

https://doi.org/10.15517/rev.biol.trop..v71i1.49004

BOTANY AND MYCOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

INTRODUCCIÓN

The Caatinga is a tropical dry forest exclu-

sively found in the Northeast of Brazil that

exhibits high biodiversity, including endemic

species. This biome covers about 845 000 km2

and is characterized by semi-arid climate, stony

and shallow soils, and deciduous vegetation

(Pinheiro & Nair, 2018). The Caatinga popula-

tion includes approximately 28 million inhab-

itants and more than 40 % are small farmers

who depend on natural resources for their

livelihoods. Unfortunately, anthropic activities

have severely disturbed the Caatinga vegeta-

tion, altering its structure, composition, and

diversity (Specht et al., 2019). This scenario

requires efforts focused on the restoration of

degraded areas.

One of the main limitations for the res-

toration of tropical dry forests is the lack of

knowledge on seed germination and seedling

establishment of strategic species (Soriano et

al., 2013). Seed germination consists of the

resumption of embryo metabolism and growth,

while seedling establishment involves the tran-

sition from heterotrophic metabolism to pho-

tosynthetic autotrophy (Bewley et al., 2013).

These developmental transitions rely on the

reserves deposited in the embryo axis and the

storage tissues as sources of metabolic ener-

gy and building blocks (Gommers & Monte,

2018). Therefore, reserve mobilization plays

a central role in seedling survival and fitness

under natural conditions (Soriano et al., 2013).

Starch, oils, and storage proteins are

reserves commonly accumulated in the stor-

age tissues during seed development (Bew-

ley et al., 2013). In desiccated seeds, these

reserves are maintained in the form of insoluble

aggregates within different cell compartments.

RESUMEN

Movilización de reservas y metabolitos secundarios durante la germinación de semillas

y el establecimiento de plántulas del árbol Erythrina velutina (Fabaceae)

Introducción: La falta de conocimiento sobre la germinación de semillas y el establecimiento de plántulas es una

de las principales limitaciones para la restauración de áreas degradadas, incluido el bosque seco tropical conocido

como Caatinga.

Objetivo: Evaluar la movilización de reservas y metabolitos secundarios durante estas etapas de desarrollo en

Erythina velutina.

Métodos: Las semillas fueron escarificadas, desinfectadas, embebidas, sembradas entre toallas de papel e incuba-

das bajo condiciones controladas. Cultivamos las plántulas hidropónicamente en un invernadero. Recolectamos

los cotiledones en la imbibición de la semilla, la protrusión de la radícula, la emergencia del hipocótilo, la for-

mación del gancho apical y la expansión de las hojas cordiformes, la primera y segunda hoja trifoliada.

Resultados: Las semillas contenían 20 % de almidón, 14.5 % de proteínas de almacenamiento, 11.6 % de lípidos

neutros y 5.7 % de azúcares no reductores en peso seco. Los azúcares solubles se consumieron desde la emergencia

del hipocótilo hasta la formación del gancho apical. Las principales reservas se movilizaron desde la formación del

gancho apical hasta la expansión de la primera hoja trifoliada. La actividad enzimática aumentó desde la mitad

hasta el final del establecimiento de las plántulas, movilizando almidón, aceites y proteínas. Se detectaron deriva-

dos de terpenoides, flavonoides, ácidos fenólicos y alcaloides. Los flavonoides y los ácidos fenólicos estuvieron en

casi todas las etapas y los derivados terpenoides desaparecieron en la expansión de las hojas cordiformes.

Conclusión: Los azúcares solubles apoyan el crecimiento temprano de las plántulas; el almidón, los aceites y

las proteínas se movilizan simultáneamente desde el establecimiento medio hasta el final por amilasas, lipasas

y proteasas ácidas. Los cotiledones contienen metabolitos secundarios, que pueden actuar en la defensa de las

plántulas. El alto contenido de reservas y los metabolitos secundarios en los cotiledones podría permitir que las

plántulas de E. velutina toleren estrés, validando su uso en la restauración de áreas degradadas.

Palabras clave: Caatinga; transición heterotrofia-autotrofia; enzimas de degradación de reservas; metabolitos

especializados; compuestos de almacenaje; leguminosa arbórea.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

When metabolism is reactivated by rehydra-

tion, reserves are degraded to generate solu-

ble metabolites, which are transported to the

embryo axis to support growth (Gommers

& Monte, 2018). Despite the importance of

this process for forest dynamics (Soriano et

al., 2013), reserve mobilization is still poorly

known in woody species, especially those from

tropical dry forests.

The mobilization process is characterized

only in a few woody species native to the Caat-

inga, like Caesalpinia pyramidalis (Dantas et

al., 2008a), Schinopsis brasiliensis (Dantas et al.,

2008b) and Anacardium occidentale (Voigt et

al., 2009). Additionally, several studies focusing

on reserve mobilization in species from other

forest biomes do not clarify the involvement

of hydrolytic enzymes in the heterotrophy-

autotrophy transition (Corte et al., 2006; Paula

et al., 2016; Veronesi et al., 2014; Weidlich et

al., 2010). Considering these knowledge limita-

tions, this work is an integrative approach on

reserve mobilization, metabolite accumulation

and hydrolase activity in the cotyledons of Ery-

thrina velutina Wild. during seed germination

and seedling establishment.

E. velutina is used as a model species for

several reasons. It is a pioneer tree legume

that naturally inhabits the Caatinga and is

recommended to the restoration of degraded

areas, since it exhibits fast growth, deep roots,

drought tolerance, and the ability to establish

symbiosis with nitrogen-fixing bacteria (Pereira

et al., 2014; Ribeiro et al., 2018; Rodrigues et

al., 2018). E. velutina also provides wood, is

an ornamental plant, and presents medicinal

properties due to the secondary metabolites it

accumulates (Rambo et al., 2019; Ribeiro et al.,

2018). Secondary metabolites include terpenes,

phenolics, and alkaloids, which are non-essen-

tial for plant growth and reproduction, but

play a part in signaling, defense, and response

to environmental stresses (Pang et al., 2021).

Although many studies have characterized

the pharmacological properties of secondary

metabolites from E. velutina, their role in seed

germination and seedling establishment are

still elusive. Considering that these compounds

may contribute to seedling survival and fitness

in natural environments (Chacón et al., 2013),

a profile of these compounds is also assessed in

parallel with reserve mobilization.

MATERIALS AND METHODS

Plant material: E. velutina seeds were har-

vested from five mother trees located at Acari,

RN, Brazil (6°27’45’’ S & 36°38’31’’ W), pack-

aged in paper bags and kept under refrigera-

tion. Seeds were mechanically scarified in the

region opposite to the hilum, immersed in

commercial detergent at 1:500 (v/v) dilution for

30 s and rinsed under running tap water. Next,

seeds were surface-sterilised with 70 % (v/v)

ethanol for 30 s followed by 0.25 % (v/v) NaClO

for 3 min, washed three times and imbibed in

sterile distilled water for 2 h. Seeds were sown

between towel paper sheets moistened with 2.5

mL of distilled water per g of dry paper. Paper

sheets were rolled up and covered with trans-

parent plastic bags. Paper rolls were maintained

upright under controlled conditions (photosyn-

thetic photon flux density of 80 μmol m-2 s-2, 12

h light/12 h dark photoperiod and 27 ± 2 °C)

for 9 days (International Seed Testing Associa-

tion, 2006).

Seven-day-old seedlings were transferred

to plastic pots containing distilled water and

were cultivated in a greenhouse for 8 days. In an

attempt to assess how the heterotrophy-autot-

rophy transition takes place when the seedlings

rely only on their reserves, nutrient solution was

not used in the hydroponic system. Seedlings

exhibiting apparent nutrient deficiencies or

morphological abnormalities were eliminated.

The harvests were performed at seven different

morphological stages (Fig. 1): imbibed seed

(stage I), radicle protrusion (stage II), hypocot-

yl emergence (stage III), apical hook formation

(stage IV), and expansion of cordiform leaves

(stage V), first trifoliate leaf (stage VI) and sec-

ond trifoliate leaf (stage VII). At every harvest,

the cotyledons were weighed and frozen at -20

°C until biochemical quantifications.

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Biochemical quantifications: Neutral lip-

ids were quantified by the gravimetric method

(Soxhlet, 1879). Samples containing 300 mg

of dry cotyledons were extracted with 8 mL

of n-hexane at 60 °C for 5 h under intermit-

tent stirring. The extracts were collected and

transferred to tubes whose mass was previously

weighed. After the evaporation of n-hexane

at 80 ºC, the mass of neutral lipids was calcu-

lated as the difference between the initial and

final mass of the tubes and expressed as mg

cotyledon-1.

To obtain an extract enriched in storage

proteins, samples of 300 mg of frozen cotyle-

dons were extracted by maceration with 1.5

mL of 100 mM Tris-HCl buffer pH 7.0 supple-

mented with 500 mM NaCl and 2 mM 2-mer-

captoethanol (Barros-Galvão et al., 2017). The

homogenates were centrifuged at 10 000 xg for

10 min, the supernatants were collected, and

pellets were re-extracted twice with 1 mL of the

extraction buffer, yielding 3.5 mL of extract per

sample. These procedures were performed at 4

°C. Soluble proteins were determined according

to the Bradford (1976) method, using bovine

serum albumin as standard. The content of sol-

uble proteins was expressed as mg cotyledon-1.

To extract the soluble metabolites, samples

containing 300 mg of frozen cotyledons were

fragmented and extracted with 5 mL of 80 %

(v/v) ethanol at 60 °C for 30 min. The super-

natants were collected, and the residuals were

re-extracted two times with 5 mL of 80 % (v/v)

ethanol under the same conditions, yielding

15 mL of ethanolic extract per sample. Total

soluble sugars (TSS) were measured with the

anthrone reagent (Morris, 1948; Yemm & Wil-

lis, 1954), employing a D-glucose standard

curve. Non-reducing sugars (NRS) were quan-

tified by a modification of the anthrone method

(Van Handel, 1968), using sucrose as standard.

Total free amino acids (TFAA) were determined

by the ninhydrin reagent (Yemm & Cocking,

1955), utilizing a L-glutamine standard curve.

The contents of all these metabolites were

expressed as μmol g-1 dry weight (DW).

Starch was extracted from the residuals

obtained after the removal of ethanol-soluble

metabolites. The residuals were macerated with

1.5 mL of 30 % (v/v) HClO4 and the homog-

enates were centrifuged at 10 000 xg for 10

min. The supernatants were collected, whereas

the pellets were re-extracted twice with 1 mL

of 30 % (v/v) HClO4, yielding 3.5 mL of extract

per sample. These steps were carried out at 4

Fig. 1. Morphological stages of Erythrina velutina during seed germination and seedling establishment: A. Seed imbibition.

B. Radicle protrusion. C. Hypocotyl emergence. D. Apical hook formation. E. Expansion of cordiform leaves. F. Expansion

of first trifoliate leaf. G. Expansion of second trifoliate leaf.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

°C. Starch was measured with the anthrone

reagent (Morris, 1948; Yemm & Willis, 1954),

using D-glucose as a standard. The values

were multiplied by 0.9 (McCready et al., 1950)

and the content of starch was expressed as

mg cotyledon-1.

Enzymatic activities: The extraction of

amylases was carried out by maceration of 300

mg of frozen cotyledons with 1.5 mL of 100

mM potassium acetate buffer pH 6.0 containing

5 mM CaCl2. The samples were centrifuged at

10 000 xg for 20 min and the supernatants were

employed as sources of enzymes. These steps

were carried out at 4 °C. Amylase activity was

assayed by the release of reducing sugars from

soluble starch (Elarbi et al., 2009). The concen-

tration of reducing sugars in the reaction medi-

um was quantified by the 3,5-dinitrosalicylic

acid method (Miller, 1959), using a D-glucose

standard curve. The activity was expressed as

μmol reducing sugars g-1 DW min-1.

Lipases were extracted from samples of 300

mg of frozen cotyledons, which were macer-

ated with 1.5 mL of 50 mM potassium phos-

phate buffer pH 7.5 supplemented with 0.01 %

(v/v) 2-mercaptoethanol. After centrifugation

at 10 000 xg for 20 min, the supernatants were

collected and used as enzymatic extracts. All

procedures were performed at 4 °C. Lipase

activity was estimated by the hydrolysis of

4-nitrophenyl-palmitate, generating 4-nitro-

phenol in the reaction medium (Marriot &

Northcote, 1975). The activity was expressed as

μmol 4-nitrophenol g-1 DW min-1.

For the extraction of acid proteases, 300

mg of frozen cotyledons were macerated with

1.5 mL of 50 mM Tris-HCl buffer pH 7.2 con-

taining 2 mM 2-mercaptoethanol. The homog-

enates were centrifuged at 10 000 xg for 20

min and the supernatants were utilized as a

source of enzymes. These procedures were

carried out at 4 ° C. Acid protease activity was

estimated by the liberation of amino acids from

casein (Beevers, 1968). The concentration of

free amino acids in the reaction medium was

measured with the ninhydrin reagent (Yemm

& Cocking, 1955), utilizing L-glutamine as a

standard. The activity was expressed as μmol

free amino acids g-1 DW min-1.

Thin-layer chromatography (TLC): Sam-

ples of 180 mg of frozen cotyledons were

solubilized in 1.8 mL of 80 % (v/v) ethanol and

maintained in an ultrasound bath at 45 °C for

30 min. The samples were centrifuged at 16 000

xg for 10 min at 25 °C, the supernatant was

transferred to another tube and the residual

was dried by SpeedVac for 5:30 h. The final

mass of the sample was assessed by gravimetry

and solubilized in 300 μl of 70 % (v/v) ethanol.

Samples were submitted to TLC by application

in silica gel 60GF254 normal phase chromato-

plates. The mobile phase was a mixture of ethyl

acetate, formic acid, acetic acid, and water in

the proportions of 10:1.1:1.1:2.6 (v/v/v/v). After

elution and observation under UV light at 254

nm, the staining was performed with vanillin/

sulfuric acid, Natural A and the Dragendorff

reagent (Izmailov & Schraiber, 1938).

Experimental design and statistical

analysis: During the experiment, samples of

cotyledons were randomly harvested at each

physiological stage. The experimental unit cor-

responded to one seedling. The biochemi-

cal quantifications were performed using four

replicates, whereas the qualitative analysis of

secondary metabolites was carried out using

three replicates. As the seedlings did not exhibit

synchronized growth, the harvests were not

performed at regular time intervals. Consider-

ing the physiological stages as categories, the

data were submitted to variance analysis and

the means were compared by the Tukey test at

5 % of confidence.

RESULTS

Significant contents of starch (Fig. 2A),

neutral lipids (Fig. 2B) and soluble proteins

(Fig. 2C) were detected in the seeds of E.

velutina. On a DW basis, the seeds contained

approximately 20 % starch, 11.6 % neutral lipids

and 14.5 % soluble proteins at the beginning of

the experiment. Besides these major reserves,

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

Fig. 2. Content of major reserves in the cotyledons of Erythrina velutina during seed germination and seedling establishment.

A. Starch. B. Neutral lipids. C. Soluble proteins. Columns represent means and error bars correspond to standard deviation

of four repetitions. Values marked with the same letter does not significantly differ according to the Tukey test at 5 % of

confidence.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

Fig. 3. Content of soluble metabolites in the cotyledons of Erythrina velutina during seed germination and seedling

establishment. A. Total soluble sugars. B. Non-reducing sugars. C. Total free amino acids. Columns represent means and

error bars correspond to standard deviation of four repetitions. Values marked with the same letter does not significantly

differ according to the Tukey test at 5 % of confidence.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

the seeds also presented a minor content of

NRS (Fig. 3B), equivalent to 5.7 % of the DW.

The content of starch (Fig. 2A) in the

imbibed seeds (stage I) was 40.7 mg cotyledon-1

and it was not significantly changed until apical

hook formation (stage IV). Starch was intensely

mobilized in the cotyledons from stage IV to

the expansion of the cordiform leaves (stage

V) since its content decreased 64 % during this

period. From then on, starch was gradually

mobilized, as its content was 14.4 mg cotyle-

don-1 at stage V and it dropped to 9.1 mg coty-

ledon-1 at the expansion of the second trifoliate

leaf (stage VII).

The content of neutral lipids (Fig. 2B) and

soluble proteins (Fig. 2C) in the imbibed seeds

was 24.1 and 31.5 mg cotyledon-1, respectively,

and it remained almost unaffected until apical

hook formation. The mobilization of oils in the

cotyledons was clearly verified from stage IV to

the expansion of the first trifoliate leaf (stage

VI), as the content of neutral lipids decreased

82.4 % during this period (Fig. 2B). Like the

mobilization of starch, the hydrolysis of storage

proteins in the cotyledons was intensified from

stage IV to V and then occurred gradually until

stage VII. Indeed, the content of soluble pro-

teins decreased 73 % from the first harvest to

the expansion of the cordiform leaves and only

3.2 % of the initial content remained at the last

harvest (Fig. 2C).

Although the mobilization of the major

carbon reserves (starch and oils) was evidenced

at stage V (Fig. 2A, Fig. 2B), the utilization

of soluble sugars in the cotyledons took place

earlier, at stage IV. In the imbibed seeds, the

content of TSS (Fig. 3A) and NRS (Fig. 3B) was

558.5 and 172.3 μmol g-1 DW, in that order, and

it was not significantly altered until stage III.

However, the content of TSS and NRS in the

cotyledons decreased 50 and 69.5 %, respec-

tively, from hypocotyl emergence to apical hook

formation. It is noteworthy that the content of

these metabolites remained unchanged in the

cotyledons during the mobilization of starch

(Fig. 2A) and oils (Fig. 2B).

Different from soluble sugars, the content

of TFAA increased in the cotyledons during

seed germination and seedling establishment

(Fig. 3C). The content of TFAA was 2.37 μmol

g-1 DW in the imbibed seeds and it increased

2.6 times at radicle protrusion (stage II). Coin-

cidently, TFAA were accumulated when the

hydrolysis of storage proteins was intensified

(Fig. 2C). In fact, the content of these metabo-

lites increased 3.7 times from stage II to V and

decreased 27 % from stage V to VII (Fig. 3C).

The activity of amylases (Fig. 4A), lipases

(Fig. 4B) and acid proteases (Fig. 4C) in the

cotyledons increased during the experiment,

especially when starch (Fig. 2A), oils (Fig. 2B)

and storage proteins (Fig. 2C) were intensely

mobilized. In the first harvest, the amylase

activity was 11.5 μmol g-1 DW min-1, it doubled

from stage I to V and a 2.3-fold increase was

noticed from stage V to VII (Fig. 4A). The

lipase activity was 22.4 μmol g-1 DW min-1 in

the imbibed seeds, it oscillated from stage I

to IV and it increased 4.5 times from stage IV

to VII (Fig. 4B). By contrast, the acid protease

activity was only 0.068 μmol g-1 DW min-1

at the beginning of the experiment, a 14-fold

increase was verified from stage I to VI and it

decreased 48 % from stage VI to VII (Fig. 4C).

When the ethanolic extracts obtained from

cotyledons were submitted to TLC, it was pos-

sible to verify that the profile of secondary

metabolites changed during seed germination

and seedling establishment (Fig. 5). Vanillin/

sulfuric acid staining suggested the occurrence

of terpenoid-derivative metabolites (brown

dots) in the seeds at Rf 0.21-0.27 and these

metabolites were detected mainly until api-

cal hook formation (Fig. 5A). According to

Natural A staining, flavonoids (yellow dots)

and phenolic acids (blue dots) were present in

almost all stages, but chlorophylls (red dots)

were detected only from stage V to VII. (Fig.

5B). The Dragendorff reagent indicated that

the seeds also contained alkaloids (orange

dots) (Fig. 5C). Curiously, an alkaloid (RF:

0.4 (I)) disappeared at radicle protrusion and

was slightly detected at hypocotyl emergence

and the expansion of the cordiform leaves.

Otherwise, an alkaloid spot was detected in all

samples near to application point (Rf < 0.1) and

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Fig. 4. Activity of hydrolytic enzymes in the cotyledons of Erythrina velutina during seed germination and seedling

establishment. A. Amylases. B. Lipases. C. Acid proteases. Columns represent means and error bars correspond to standard

deviation of four repetitions. Values marked with the same letter does not significantly differ according to the Tukey test at

5 % of confidence.

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Fig. 5. Thin-layer chromatograms of ethanolic extracts obtained from the cotyledons of Erythrina velutina during seed

germination and seedling establishment. A. Staining with vanillin/sulfuric acid for terpenoid-derivatives [brown dots-*Rf:

0.21 (I, III, IV), **Rf: 0.27 (II)] and other steroids (violet dots). B. Staining with Natural A for flavonoids (yellow dots),

phenolic acids (blue dots), and chlorophylls (red dots), and C. Staining with the Dragendorff reagent for alkaloids [orange

dots-#Rf: 0.40 (I)].

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

it remained throughout seed germination and

seedling establishment. The exact number of

alkaloids in this spot and their chemical identi-

ties remain to be investigated.

DISCUSSION

The seeds of tree legumes native to the

Caatinga are still poorly characterized in terms

of nutritional reserves and only a few efforts

have been made to evaluate the potential use

of these seeds as food, feed, and industrial raw

material (Carvalho et al., 2011; Mayworm et

al., 1998). Edible legume seeds are commonly

classified into pulses, which are almost devoid

of lipids, and oilseeds, which are poor in starch

(Vijayakumar & Haridas, 2021). As E. velu-

tina seeds contain approximately 20 % starch,

11.6 % oils and 14.5 % storage proteins in a

DW basis, they cannot be classified according

to these categories. The seeds of other tree

legumes also demonstrate balanced contents

of starch and oils, like Caesalpinia bracteosa

and Pterogyne nitens (Carvalho et al., 2011).

Although the content of storage proteins in

E. velutina seeds is similar to that found in

Poecilanthe ulei (Mayworm et al., 1998) and

C. bracteosa (Carvalho et al., 2011), the seeds

of several tree legumes native to the Caatinga

contain more than 30 % storage proteins (Car-

valho et al., 2011; Mayworm et al., 1998). The

diversity and proportion of major reserves

stored by E. velutina seeds may be related to

metabolic flexibility during the heterotrophy-

autotrophy transition.

In addition to the major reserves, E. velu-

tina seeds also contain nearly 5.7 % NRS

on a DW basis. These compounds generally

include sucrose and raffinose family oligo-

saccharides, which are considered as minor

reserves. NRS may contribute to the longevity

of orthodox seeds by stabilizing the glassy state

and the membrane structure under desicca-

tion (Bewley et al., 2013) and they probably

play a role as immediate sources of respiratory

substrates during seed germination (Rosental

et al., 2014). The content of NRS in E. velutina

seeds is comparable to that verified for other

species, which varies from 2 to 6 % of the DW

(Bewley et al., 2013).

As a pioneer species, E. velutina produces

high quantity of large and viable seeds every

year (Ribeiro & Dantas, 2018). In this work, it

is verified that approximately 52 % of the seed

DW correspond to nutritional reserves. There-

fore, the size and reserve content of E. velu-

tina seeds may be functional traits, since it is

expected that large seeds containing abundant

reserves are able to produce vigorous seedlings,

which are likely to growth and survive under

adverse conditions (El-Keblawy et al, 2018).

Considering that environmental stresses such

as water deficit and high temperature are com-

monly found in the Caatinga (Pinheiro & Nair,

2018), E. velutina may take advantage of these

seed functional traits to enable successful seed-

ling establishment.

As the content of starch (Fig. 2A), neutral

lipids (Fig. 2B), and soluble proteins (Fig. 2C) in

the cotyledons remain almost unaffected from

seed imbibition (stage I) to radicle protrusion

(stage II), there is no clear evidence that the

mobilization of major reserves starts during the

germination of E. velutina seeds. These results

corroborate the notion that the mobilization

of major reserves is a post-germinative event

(Bewley et al., 2013). Like E. velutina, the mobi-

lization of oils and storage proteins starts after

seed germination in Caesalpinia peltophoroides

(Corte et al., 2006), the content of soluble pro-

teins remains unchanged in germinating seeds

of Aniba rosaeodora (Lima et al., 2008) and

starch mobilization is not detected during seed

germination in Enterolobium contortisiliquum

(Veronesi et al., 2014). Although the reserves

stored in the embryo axis are not measured in

this study, it is likely that they have sustained

the germination of E. velutina seeds. In some

species, the embryo axis stores low contents

of sucrose, raffinose family oligosaccharides,

oils, and storage proteins. It is accepted that the

hydrolysis of these reserves during metabolism

reactivation provides substrates for respiration

and amino acids for protein synthesis (Bewley

et al., 2013; Rosental et al., 2014).

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

In the cotyledons of E. velutina seedlings,

the carbon and nitrogen reserves are simultane-

ously mobilized from mid to late establishment

(Fig. 2), since the degradation of starch (Fig.

2A), oils (Fig. 2B), and storage proteins (Fig.

2C) is intensified from apical hook formation

to the expansion of the first trifoliate leaf. It is

well established that the hexoses released by

starch degradation (Dong & Beckles, 2019)

and the fatty acids resulting from oil hydrolysis

(Theodoulou & Eastmond, 2012) are converted

to sucrose for export to the seedling axis. It

is also clear that the amino acids liberated by

storage protein hydrolysis support protein syn-

thesis in the storage tissues and are converted

to amides for export to the growing tissues

(Bewley et al., 2013). However, the metabolic

processes responsible for the mobilization of

different reserves in the cotyledons of E. velu-

tina seedlings may be interdependent, consid-

ering that they occur simultaneously. Previous

reports demonstrate that fatty acids and amino

acids are alternative respiratory substrates and

carbon skeletons derived from fatty acid deg-

radation may be used for amino acid synthesis

during the heterotrophy-autotrophy transition

(Borek et al., 2015; Hildebrandt et al., 2015).

If this metabolic flexibility occurs in E. velu-

tina, it possibly contributes to seedling fitness

under stresses.

It is noteworthy that the utilization of

soluble sugars precedes the mobilization of

major reserves in the cotyledons of E. velu-

tina seedlings. As the content of TSS (Fig. 3A)

and NRS (Fig. 3B) decreases from stage III to

IV, soluble sugars may have supported early

seedling establishment. Accordingly, NRS like

sucrose and raffinose family oligosaccharides

are more accessible reserves than starch and oils

(Rosental et al., 2014). In the woody species Pel-

tophoprum dubium (Veronesi et al., 2014) and

Morinda citrifolia (Paula et al., 2016), seedling

growth also initiates at the expense of soluble

sugars provided by the storage tissues.

The mobilization of starch (Fig. 2A) and

oils (Fig. 2B) does not result in accumulation

of soluble sugars (Fig. 3A and B) in the cotyle-

dons of E. velutina seedlings from mid to late

establishment. Similarly, the mobilization of

carbon reserves does not increase the content

of soluble sugars in the cotyledons during

seedling establishment in the tree legumes C.

peltophoroides (Corte et al., 2006) and Schizolo-

bium parahyba (Weidlich et al., 2010). As a

developmental transition, seedling establish-

ment involves alterations in the source-sink

relationship, since the growth of new organs

generates additional carbon sinks (Wingler,

2018). In E. velutina seedlings, the axis sink

strength may have predominantly increased at

stage V, when the expansion of the cordiform

leaves coincides with the growth of secondary

roots. Given that soluble sugars are the major

products of starch and oil degradation (Dong &

Beckles, 2019; Theodoulou & Eastmond, 2012),

these metabolites may have been translocated

to the seedling axis to support its growth.

The mobilization process involves the

coordinated action of several enzymes inside

different cell compartments in the storage tis-

sues. Amylases are essential for the dismantling

of starch granules in amyloplasts (Bewley et al.,

2013), lipases are responsible for the hydrolysis

of triacylglycerols in oleosomes (Borek et al.,

2015) and acid proteases catalyse the first cleav-

ages of protein aggregates in storage vacuoles

(Tan-Wilson & Wilson, 2012). In the cotyledons

of E. velutina seedlings, the activity of amylases

(Fig. 4A), lipases (Fig. 4B) and acid proteases

(Fig. 4C) simultaneously increases from mid to

late establishment, enabling the mobilization

of starch (Fig. 2A), oils (Fig. 2B), and storage

proteins (Fig. 2C). Although only a few works

have evaluated the activity of hydrolases during

reserve mobilization in other woody species,

the activity of amylases and acid proteases also

increases after seed germination in A. rosaeo-

dora (Lima et al., 2008) and Pistacia vera (Einali

& Valizadeh, 2017), respectively. Curiously, an

increase in the activity of reserve-degrading

enzymes (Fig. 4) coincides with a decrease in

the content of soluble sugars (Fig. 3A, Fig. 3B)

and an accumulation of TFAA (Fig. 3C) in the

cotyledons of E. velutina seedlings. Considering

that carbon availability is involved in the regu-

lation of seedling metabolism (Wingler, 2018),

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

it is possible that decreased sugar supply plays

a part in inducing the activity of hydrolases.

Additionally, amino acid accumulation could

be related to enhanced protein turnover in the

cotyledons (Tan-Wilson & Wilson, 2012).

Interestingly, the profile of secondary

metabolites in the cotyledons of E. velutina

changes during seed germination and seedling

establishment (Fig. 5). As previously reported

for the genus Erythrina (Rambo et al., 2019),

terpenoid-derivatives (Fig. 5A), flavonoids,

phenolic acids (Fig. 5B), and alkaloids (Fig. 5C)

are detected by TLC. Even though the roles of

secondary metabolites in early development are

still poorly investigated, it is proposed that they

are involved in seedling defence (Pang et al.,

2021). Since flavonoids and phenolic acids are

found at almost all stages (Fig. 5B), they might

play a part as a constitutive defence against

pathogens (Chacón et al., 2013). By contrast,

terpenoid-derivatives are detected only during

seed germination and early seedling establish-

ment (Fig. 5A). The universal stain vanillin/

sulfuric acid used in TLC does not reveal the

exact class of terpenoid-derivatives present in

the samples. Among these compounds, the

occurrence of saponins and steroids may be

the most plausible. Like flavonoids, terpenoid-

derivatives could also be involved in seedling

defence as they demonstrate antifungal activity

(Naboulsi et al., 2018).

In addition to the secondary metabolites

mentioned above, an alkaloid dot is found

throughout the experiment, while another

disappears at radicle protrusion (Fig. 5C). It

has recently been demonstrated the occur-

rence of erythraline and erysodine in the seeds

used herein, which are alkaloids found in the

embryo, testa and mainly in the cotyledons

(unpublished data). Considering that major

reserves (Fig. 2) and soluble sugars (Fig. 3A,

Fig. 3B) are not degraded, TFAA are accumu-

lated (Fig. 3C), and some alkaloids disappear

(Fig. 5C) in the cotyledons of E. velutina during

seed germination, these alkaloids might play a

role as nitrogen reserves during seed germina-

tion (Chacón et al., 2013). Alternatively, they

could also exhibit allelopathic activity (Oliveira

et al., 2012), contributing to seedling fitness

during early establishment.

According to the results, it is possible to

conclude that carbon and nitrogen reserves

stored in the cotyledons of E. velutina are mobi-

lized after seed germination. Soluble sugars

support early seedling growth, while starch,

oils, and storage proteins are utilized from mid

to late establishment. Major reserves are simul-

taneously mobilized by the coordinated action

of amylases, lipases, and acid proteases, espe-

cially during the expansion of the first leaves.

E. velutina seeds contain terpenoid-derivatives,

flavonoids, phenolic acids, and alkaloids, which

may play a part in seedling defense during

germination and establishment. The high con-

tent of reserves and the presence of secondary

metabolites in the cotyledons could provide E.

velutina seedlings with the ability to survive

stresses, supporting their use in the restoration

of degraded areas.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

This work was supported by Coordena-

ção de Aperfeiçoamento de Pessoal de Nível

Superior (fellowship); Universidade Federal do

Rio Grande do Norte (fellowship); São Paulo

Research Foundation (FAPESP grant INCT-

BioNat 2014/50926-0) and Conselho Nacional

de Desenvolvimento Científico e Tecnológico

(Brazil) (grant INCT BioNat 465637/2014-0).

The authors acknowledge Daisy Sotero Chacon

for the support to carry out the TLC procedures.

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e49004, enero-diciembre 2023 (Publicado Ago. 24, 2023)

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