South African Journal of Botany 97 (2015) 149153

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Analysis of nitrogen seed reserves of ten tree species of the

tropical dry forest
Diana Soriano 1,2, Sandra Alvarado-Lpez, Esther Ziga-Snchez, Alma Orozco-Segovia, Alicia Gamboa-deBuen
Laboratorio de Ecologa Fisiolgica, Departamento de Ecologa Funcional, Instituto de Ecologa, Universidad Nacional Autnoma de Mxico, Mxico D.F. 04510, Mexico

a r t i c l e

i n f o

Article history:
Received 29 March 2014
Received in revised form 14 December 2014
Accepted 5 January 2015
Available online 4 February 2015
Edited by PN Hills
Keywords:
Seeds
N
Heat-stable proteins
Amino acids
Tropical dry forest

a b s t r a c t
Nitrogen is a component of many seed reserve compounds, which play a remarkable role during germination and
have great value for predators. A predened coefcient factor is used to determine the protein concentration
from the seed nitrogen concentration. However, this calculation may not reect the variety of nitrogen compounds present in seeds and their ecological role. Understanding how the seed nitrogen content is distributed between amino acids and proteins will help explain how nitrogen compounds are involved in seed metabolism. We
studied the heat-stable protein and total amino acid concentrations in ten tree species in a tropical dry forest in
northwest Mexico. The seed nitrogen concentrations formed a continuum in the studied species. The total amino
acid concentration was positively correlated with the nitrogen concentration. During germination, a differential
decrease in the nitrogen, total amino acid, and heat-stable protein contents was detected among the species.
Functional diversity in the partitioning of nitrogen inside seeds is discussed.
2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction
Functional diversity reects ecological and evolutionary strategies
used by species, and these strategies are related to temporal variations
in the availability and partitioning of resources, which is a fundamental
factor underlying species coexistence. Functional diversity in plants is
expressed by the variation of seed biomass, seed reserve composition,
and germination variables. Seed reserve composition among tropical
species is highly variable (Soriano et al., 2011). The main seed reserves
are carbohydrates (structural and non-structural), lipids, and nitrogen
compounds. Nitrogen is a component of many compounds, including
proteins, amino acids, nucleic acids, amides, and ammonium salts
(Ezeagu et al., 2002). Additionally, N content has an important ecological role because it is involved in seed predation, germination, and
seedling success (Lieffering et al., 1996; Grubb et al., 1998; Hara and
Toriyama, 1998; Soriano et al., 2013).
Two of the most important N compounds, proteins and amino acids,
are essential during germination and seedling growth. During germination, the storage proteins are mobilised into soluble peptides and free
amino acids, and then, these compounds are translocated to the embryonic axis to provide energy (Mayer and Poljakoff-Mayber, 1982;

E-mail address: diana.soriano.fernandez@usherbrooke.ca (D. Soriano).

Tel.: +52 55 46229008.
Present address: Facult des Sciences. Dpartement de Biologie. Universit de
Sherbrooke. 2500, boul. de l'Universit, Sherbrooke, Qubec J1K 2R1E, Canada. Tel.: +52
819 821 8000, poste 66578.
1
2

http://dx.doi.org/10.1016/j.sajb.2015.01.003
0254-6299/ 2015 SAAB. Published by Elsevier B.V. All rights reserved.

Mntz et al., 2001; Schlereth et al., 2000). In germinating seeds, amino

acids are a carbon source for precursors of key metabolites involved in
the tricarboxylic acid cycle and sugar synthesis. Amino acids can also
be used as protein building blocks (Gallardo et al., 2001).
Typically, in ecological studies, total seed N concentration is determined and converted to protein concentration using a predened coefcient factor. However, this factor overestimates seed protein
concentration and cannot reect important differences in N distribution
or N's rate of use and fate (Grubb et al., 1998; Soriano et al., 2013). This is
because non-protein compounds constitute a considerable proportion
of the N content (Tkachuk, 1969). Non-protein N compounds that are
present in seeds include secondary compounds such as alkaloids, amides, free amino acids, and non-protein amino acids (McKey, 1974;
Rhoades and Coates, 1976). This diversity of non-protein compounds
is especially important in seeds from wild species, in which these compounds might have an important ecological role (Ezeagu et al., 2002).
For example, legume seedlings accumulate non-protein amino acids
and metabolic intermediates in amino acid biosynthesis or in other
metabolic pathways (Martnez-Villaluenga et al., 2006).
In seeds, reserve proteins are the most abundant. High levels of
reserve proteins are present during seed maturation and desiccation
and represent an important fraction of the named heat-stable proteins
(Gallardo et al., 2001). To determine the differential role of amino
acids and heat-stable proteins in seed germination, we studied ten
tree species from ve different families in a tropical dry forest (TDF) in
northwest Mexico. Using dry and germinated seeds, we determined
the N, heat-stable protein and the total amino acid concentrations.

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D. Soriano et al. / South African Journal of Botany 97 (2015) 149153

Table 1
List of studied species, family and average dry mass (mg) of reserves tissues of control
(non-germinated) and germinated seeds (n = 30 seeds, means s.e.).
Dry mass (mg)
Species

Family

Control

Germinated

1
2
3
4
5
6
7
8
9
10

Fabaceae
Fabaceae
Fabaceae
Fabaceae
Fabaceae
Fabaceae
Hernandiaceae
Rubiaceae
Polygonaceae
Meliaceae

36.3 0.8
21.2 0.5
3.4 0.2
158.9 2.1
146.1 2.9
539.1 11.9
190.1 5.6
1.2 0.2
17.1 0.5
431.2 19.2

27.5 1.2
13.3 1.0
2.8 0.02
148.5 6.5
121.3 6.0
513.7 31.6
188.1 6.5
1.0 0.01
15.1 1.1
416.8 24.2

Acacia farnesiana
Acacia sp.
Apoplanesia paniculata
Caesalpinia eriostachys
Caesalpinia platyloba
Enterolobium cyclocarpum
Gyrocarpus jatrophifolius
Hintonia latiora
Ruprechtia fusca
Swietenia humilis

protein patterns for each sample were determined with previously

obtained heat-stable protein extract by separation in a denaturing 12%
acrylamide gel (SDS-PAGE) and staining with Coomassie blue. To determine the free amino acid concentration, we used 100 mg samples for
each replicate, following the Ninhydrin method (Rosen, 1957). N,
heat-stable protein, and amino acid reserve concentrations were reported as a percentage of the reserve tissue dry mass.
2.4. Statistical analysis
We performed regression analysis between the concentrations of N,
and heat-stable proteins and amino acids in non-germinated seeds.
Regression analyses were made using TableCurve 2D, v3 (AISN Software, Chicago, IL, USA). To compare N, amino acid and heat stable

Additionally, we obtained the protein pattern for ten tree species. We

asked the following questions. (1) How is N concentration distributed
between heat-stable proteins and amino acids? (2) Is there a relationship between N concentration and protein or amino acid concentration?
2. Material and methods
2.1. Plant material
Seeds from the ten studied species (Table 1) were collected from
areas surrounding the Biological Station of Chamela (19 30 N, 105
30 W) on the Pacic coast of Mexico. The main vegetation in this region
is the tropical dry forest (TDF). The mean annual temperature is 24.9 C,
and the mean annual precipitation is 788 mm (19772003) at this site.
On average, 80% of the annual precipitation occurs between July and
October (Bullock and Sols-Magallanes, 1990). Seeds of all the studied
species germinate at the beginning of the growing season (Soriano
et al., 2014). Collected seeds were stored in plastic containers in semicontrolled conditions until prior to the germination experiment. These
seeds were also used as control seeds. The dry mass of the seeds highly
differs among the studied species (from 1085 mg Enterolobium
cyclocarpum seeds to 1.6 mg seeds of Hintonia latiora; Soriano
et al., 2011).
2.2. Germination experiments
To perform the germination experiments, the seeds were germinated in agar 1% plates and then placed into growth chambers (Lab-Line Instruments, Inc., Melrose Park, IL) with a photoperiod of 12/12 h light/
dark at 25 C, photon ux density (400700 nm) of 33 mol m2 s1,
and RH of 3547%. Germination was characterised by 2 mm of radicle
protrusion. Prior to seed incubation, the species with impermeable
seeds (Acacia farnesiana, Acacia sp., Caesalpinia platyloba, and
E. cyclocarpum) were scaried with 98% H2SO4 to obtain synchronous
germination. Once germinated, we isolated the radicle and testa from
cotyledons and extra-embryonic tissues (reserve tissues). The reserve
tissues were frozen in liquid N to later reserve determination.
2.3. Reserve determination
We determined N, heat-stable protein, and amino acid concentrations in the reserve tissues of control and germinated seeds. We conducted three replicates for each determination, and each sample was
obtained from at least 10 seeds. For N determination we used samples
of 10 mg from each species. N concentrations were quantied with an
elemental analyser (NC 2500; Thermo Quest S.P.A., Rodanao, Milan,
Italy). Heat-stable proteins were extracted and puried according to
the methods of Gonzlez-Zertuche et al. (2001). We utilised 100 mg
of seeds for each replicate. Heat-stable protein concentrations were
determined with the Bradford method (Bradford, 1976). Heat-stable

Fig. 1. A) Nitrogen concentrations, B) heat-stable protein concentrations and C) free amino

acid concentration in control seeds from the studied species. Numbers are in accordance
with Table 1.

D. Soriano et al. / South African Journal of Botany 97 (2015) 149153

Table 2
Average content (mg) of N, heat stable proteins and free amino acids in control (C, nongerminated) and germinated seeds (G). Asterisk denote signicant differences (t-test)
*p b 0.05; **p b 0.001 (n = 30, means s.e.).
N
Species
1
2
3
4
5
6

Acacia farnesiana
Acacia sp.
Apoplanesia paniculata
Caesalpinia eriostachys
Caesalpinia platyloba
Enterolobium
cyclocarpum
7
Gyrocarpus jatrophifolius
8
Hintonia latiora
9
Ruprechtia fusca
10 Swietenia humilis

3.01
2.53**
2.26
1.24**
0.18
0.20
9.51
8.06**
9.01
8.35**
42.92 38.39**

Heat-stable
proteins

Amino acids

0.99
0.52
0.02
2.47
2.01
1.33

0.30**
0.17*
0.006**
1.35**
0.99**
1.24

8.48
2.78**
7.19
1.85**
0.07
0.05**
5.27
3.41**
10.31
5.9*
138.38 58.21**

15.72 14.39** 0.61

0.31*
0.04
0.04
0.001 0.0001*
0.84
0.39* 0.11
0.06*
12.45 12.52
0.33
0.08*

151

total amino acid content with respect to the control seeds (t-test,
P b 0.05; Table 2). The decrease varied between 28.5% in A. paniculata
and 74.2% in Acacia sp.

4. Discussion
G

3.96
0.01
0.26
7.16

3.46
0.01
0.18
6.77

protein content (concentration dry mass) between control and germinated seeds from the ten studied species, we performed a t-test per each
species. Statistical analyses were made using Sigma Plot v.11 (Jandel
Scientic, Core Madera, CA).
3. Results
The seeds from the ten studied species represent a continuum of N
concentration, between 11% in Acacia sp. and 3% in Swietenia humilis
(Fig. 1A). Six of the seven species with the highest N content were
Fabaceae species. The heat-stable protein concentrations were between
2.7% in A. farnesiana and 0.2% in E. cyclocarpum (Fig. 1B) and were not
signicantly correlated with the N concentration (P N 0.05). At germination time, seven species showed a signicant reduction in N content (ttest, P b 0.05; Table 2). Meanwhile, there was a signicant decrease in
the heat-stable protein concentration in germinated seeds relative to
control seeds for nine of the ten species (Table 2; t-test, P b 0.05). This
decrease varied from 90% in H. latiora to 6.7% in E. cyclocarpum.
The protein pattern of the studied species showed differences between the control and germinated seeds. Compared with the control
seeds, the proteins that were not detected in germinated seeds or that
were only faintly detected were mostly in the 2040 kDa range
(Fig. 2). Notably, a protein band of approximately 28 kDa was absent
from A. farnesiana and Gyrocarpus jatrophifolius. No protein was detected in germinated E. cyclocarpum and H. latiora seeds or in Ruprechtia
fusca control and germinated seeds.
The total amino acid concentration was between 35.2% in Acacia sp.
and 1.5% in G. jatrophifolius (Fig. 1C). The total amino acid concentration
was positively correlated with N concentration in the control seeds
(Fig. 3). In the germinated seeds of the six Fabaceae species (A.
farnesiana, Acacia sp., Apoplanesia paniculata, Caesalpinia eriostachys, C.
platyloba and E. cyclocarpum), there was a signicant decrease in the

The study of seed reserves could provide important evidence about

seed metabolism, seedling establishment, species distribution, plant
animal interactions and potential utility as a food source (Ezeagu
et al., 2002). Examining seed N content in wild species is important because different N compounds may have distinct, specic ecological roles
(Beckman, 2013).
The heat-stable protein fraction, quantied in this study, and which
mainly contains reserve proteins, was not signicantly correlated with
the N concentration in the control seeds. Based on this result the usual
N to protein conversion factors would not reect the reserve protein
concentration in the seeds of the studied species. In this study, the N
concentration might more accurately reect the free amino acid concentration in the seed. Our results show that there is a positive relationship between free amino acid concentration and the N concentration.
The relationship between the N concentration and the germination
rate previously reported (Soriano et al., 2011) may be correlated with
the amino acid concentration in the seed. This might indicate that we
need put attention on the amino acid concentration and characterisation when we were interested on the germination process (Onomo
et al., 2010).
The four Fabaceae species (Acacia sp., A. farnesiana, E. cyclocarpum
and C. eriostachys) had the highest heat-stable protein concentrations
of all the tested species. This large amount of heat-stable protein could
be related to their importance during germination (Hara and
Toriyama, 1998) and might be linked to the success of this family
(Lott and Atkinson, 2002). Despite the widely reported mobilisation of
N compounds during germination in the studied species (Soriano
et al., 2013), the N concentration signicantly decreased in seven species on germination (Acacia sp., A. farnesiana, C. eriostachys, C. platyloba,
E. cyclocarpum, G. jatrophifolius, and R. fusca). In contrast, the heat-stable
protein concentration decreased in nine species, including three of the
previously mentioned Fabaceae species (Acacia sp., A. farnesiana and C.
eriostachys). Amino acids have a central role in germination metabolism. The nding that not all of the species showed a decrease in
amino acid concentration after germination might be related to the
inter-conversion of N compounds, particularly proteins (Nonogaki,
2008).
The decrease in the heat-stable protein content after germination
(Table 2) was in accordance with the pattern change determined by
SDS-PAGE (Fig. 2). The intensity of certain protein bands between 20
and 40 kDa decreased in all of the studied species that showed a quantitative decrease in protein concentration. Globulins, the major seed
storage proteins of dicotyledonous plants, have been detected in this

Fig. 2. Patterns of heat-stable proteins. Acrylamide gel stained with Coomassie blue. (MW) Molecular weight for control (C) and germinated (G) seeds. Numbers are in accordance with
Table 1.

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D. Soriano et al. / South African Journal of Botany 97 (2015) 149153

References

Fig. 3. Relationship between N and amino acid concentrations in the studied species:
y = 2.16 + 0.13x(2ln x).

molecular mass range in Wigandia urens (Mntz et al., 2001;

Gamboa-deBuen et al., 2006).
The large amount of N that is not present in amino acids or proteins
(N50%) in the Fabaceae species may be derived from tannins (MuellerHarvey, 2006). Tannins may serve as protection against predation,
owing to the high nutritional value of Fabaceae species (Vestena et al.,
2001; Wink, 2013). The N-containing defence compounds have a dual
function: being toxic and as nitrogen storage compounds which are
remobilized during germination (Wink, 2013). As a consequence such
can be related to the seed palatability and the transport of seeds by
predators, inuencing the dispersal and seed and seedling survival
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One of the main examples of N partitioning diversity was found in
the seeds of G. jatrophifolius. This species had the second highest N concentration of the tested species but showed low heat-stable protein and
amino acid concentrations. G. jatrophifolius seeds are not predated in
eld conditions in a previous two year study (Soriano et al., 2014),
which indicates that N is allocated to secondary compounds, possibly
alkaloids or tannins (Payne and Foley, 1992; Sotelo et al., 1993). This
has been described previously in Erytrina species, where the amino
acid content is usually in inverse proportion to the alkaloid content
(Garca-Mateos et al., 2005). The functional diversity of the N compounds needs to be studied carefully because of their direct impact on
plant species tness (Wink, 2013).
Our results can have both methodological and theoretical implications in the study of seed N compounds in wild species. Our data
showed that the N concentration diversity extends to the partitioning
and utilisation of N inside the seeds. Even if Fabaceae species contained
more N than the other tested species, there was no clear pattern of
mobilisation. The N compounds that were mobilised by most of the
studied species were proteins, demonstrating the importance of proteins during germination. The observed differences in the patterns of
N mobilisation among the studied species show that plants have different mechanisms for germination and seedling growth.

Acknowledgements
We thank Chamela Biology Station (IB-UNAM) for the use of their
research facilities. We thank Irma Acosta Calixto for their technical
assistance. This work was supported by Direccin General de
Asuntos del Personal Acadmico-UNAM [IN 201912] and Consejo
Nacional de Ciencia y Tecnologa [postdoctoral fellowship to Diana
Soriano 3910].

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