Revista Fitotecnia Mexicana
ISSN: 0187-7380
revfitotecniamex@gmail.com
Sociedad Mexicana de Fitogenética, A.C.
México
Castañeda-Saucedo, M. Claudia; Córdova-Téllez, Leobigildo; Tapia-Campos, Ernesto; DelgadoAlvarado, Adriana; González-Hernández, Víctor A.; Santacruz-Varela, Amalio; Loza-Tavera, Herminia;
García-de-los-Santos, Gabino; Vargas-Suárez, Martín
DEHYDRINS PATTERNS IN COMMON BEAN EXPOSED TO DROUGHT AND WATERED
CONDITIONS
Revista Fitotecnia Mexicana, vol. 37, núm. 1, 2014, pp. 59-68
Sociedad Mexicana de Fitogenética, A.C.
Chapingo, México
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Rev. Fitotec. Mex. Vol. 37 (1): 59 - 68, 2014
Artículo Científico
DEHYDRINS PATTERNS IN COMMON BEAN EXPOSED TO DROUGHT AND WATERED CONDITIONS
PATRONES DE DEHIDRINAS EN PLANTAS DE FRIJOL EXPUESTAS A SEQUÍA Y RIEGO
M. Claudia Castañeda-Saucedo1*, Leobigildo Córdova-Téllez1, Ernesto Tapia-Campos3,
Adriana Delgado-Alvarado2, Víctor A. González-Hernández1, Amalio Santacruz-Varela1,
Herminia Loza-Tavera4, Gabino García-de-los-Santos1 and Martín Vargas-Suárez4
1
Postgrado de Recursos Genéticos y Productividad, Campus Montecillo, Colegio de Postgraduados. Km 36.5 Carretera México-Texcoco. 56230, Montecillo, Edo.
de México, México. 2Programa en Estrategias para el Desarrollo Agricola Regional, Campus Puebla, Colegio de Postgraduados. Km 125.5 Carretera Federal
México-Puebla. 72760, Puebla. *Centro Universitario del Sur, Universidad de Guadalajara. Av. Enrique Arreola Silva # 883. 49000, Col. Centro, Ciudad Guzmán,
Jalisco. Tel 341 575 22 22 Ext 46126. 3Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Normalistas 800. 44270, Colonia
Colinas de la Normal, Guadalajara, Jalisco. 4Facultad de Química, Departamento de Bioquímica, Universidad Nacional Autónoma de México. Avenida Insurgentes
Sur 3000, 04510. Coyoacán, D. F.
*Autor de correspondencia (csaucedo@colpos.mx)
SUMMARY
Drought is a major constraint for common bean (Phaseolus vulgaris
L.) production in México. Dehydrins are constitutive or stressinduced proteins related with a protective role of membranes and
macromolecules against denaturation, thus preventing loss of their
function. In this work, seed production and patterns of dehydrins
accumulation in leaves and pods were evaluated in common bean cv.
‘Otomí’ subjected to drought, as compared with well-irrigated plants.
Drought applied at pod formation and seed filling (SF) reduced yield
up to 57 %. An antibody against a consensus sequence present in most
dehydrins allowed for dehydrin identification. Two dehydrins of 82
and 73 kDA turned up both in leaves and pods throughout all the
evaluated conditions. Presumably, both dehydrins are constitutive in
the ‘Otomí’ cultivar. These dehydrins showed higher expression than
controls in leaves after 6 d of drought at seedling and SF stages, and in
pods 6 d after drought had started at SF. Increased expression might
provide better protection during early stages of seedling and seed
development. Increments on 63, 36 and 22 kDa dehydrin expression
in pods at late SF might coincide with plant developmental programs,
which prepare seed for desiccation. Dehydrins of 158, 54, 46, and 41
kDa were detected in pods 10 d after floral opening as a transient
response to drought stress in SF. These results indicate dehydrins are
relevant during plant development, as well as during drought stress.
Index words: Phaseolus vulgaris, dehydrins, drought, vegetative growth,
pod development, seed filling.
RESUMEN
La sequía es el factor más limitante de la productividad de frijol
(Phaseolus vulgaris L.) en México. Las dehidrinas son proteínas
constitutivas o inducibles por diferentes tipos de estrés, relacionadas
con la protección a membranas y macromoléculas contra la
desnaturalización, que evitan la pérdida de función. En este trabajo se
estudió el efecto de la sequía aplicada en diferentes etapas de desarrollo,
sobre la producción de semilla y los patrones de acumulación de
dehidrinas en hojas y vainas de frijol cv. ‘Otomí’, comparado con plantas
irrigadas. La sequía aplicada durante el período de formación de
vainas y llenado de semilla (SF), redujo hasta 57 % el rendimiento. Las
dehidrinas fueron identificadas con un anticuerpo que reconoce una
secuencia consenso presente en la mayoría de ellas. Dehidrinas de 82 y
73 kDa fueron detectadas en hojas y en vainas en todas las condiciones
Recibido: 9 de Febrero del 2011
Aceptado: 15 de Noviembre del 2013
evaluadas, por lo que se consideran constitutivas del cultivar ‘Otomí’.
Estas dehidrinas presentaron mayor expresión que el control en hojas
a los 6 d de sequía aplicada en los estadios de plántula y SF, y en vainas
también a los 6 d de sequía aplicada en SF. Estos incrementos podrían
ser necesarios para brindar mejor protección en etapas tempranas del
desarrollo de la plántula y la semilla. Incrementos en dehidrinas de 63,
36 y 22 kDa observados en vainas durante la etapa de SF, podrían ser
parte del programa de desarrollo de la planta encargado de preparar
a la semilla para la desecación. En vainas se detectaron dehidrinas de
158, 54, 46 y 41 kDa a los 10 d después de la apertura floral, como una
respuesta transitoria al estrés hídrico en SF. Estos resultados indican
que las dehidrinas de frijol son relevantes durante el desarrollo de la
planta y durante condiciones de estrés por sequía.
Palabras clave. Phaseolus vulgaris, desarrollo vegetativo, dehidrinas,
desarrollo de vaina, llenado de semilla, sequía.
INTRODUCTION
Drought is a major environmental stress that limits crop
productivity worldwide causing significant yield reductions (Gebeyehu et al., 2010). Under drought stress, plant
responses are diverse. For example, common bean (Phaseolus vulgaris L.) exhibits morphological plasticity characterized by over-producing reproductive structures (Acosta et
al., 2003); physiological changes, such as reduction of stomatal conductance and photosynthetic rates, have been recorded in kidney bean (Phaseolus vulgaris L.) (Miyashita et
al., 2005); increased respiration in Arabidopsis (Shinozaki
and Yamaguchi-Shinozaki, 2007) and sugar loss takes place
in Arabidopsis and wheat (Tritricum aestivum L.) (Xue et
al., 2008). At the subcellular level gene expression changes,
induced by plant protection responses against environmental stress, have also been reported in response to drought.
Some of these changes include synthesis of chaperons, dehydrins, osmotins, aquaporins, among other compounds,
which directly act on stress tolerance, and synthesis of regulatory proteins such as transcription factors, kinases and
enzymes of the phospholipids metabolism (Shinozaki and
DEHYDRIN PATTERNS IN DROUGHT STRESSED BEAN PLANTS
Rev. Fitotec. Mex. Vol. 37 (1) 2014
Yamaguchi-Shinozaki, 2007).
on small-scale farms (Jones, 1999). Beans produced by resource-poor farmers are more vulnerable to abiotic stresses,
such as drought and low soil fertility (Miklas et al., 2006).
Dehydrins are biochemically classified as Group 2 LEA
(late embryogenesis abundant) proteins (Close et al., 1989).
Some of them are ABA-inducible and synthesized in different tissues of many plant families in response to several
stresses, such as drought, freezing, salt stress, and even
heavy metals (Hanin et al., 2011). Some dehydrin proteins
play important protective roles during cellular dehydration,
but their precise function remains unclear (Tripepi et al.,
2011). It has been proposed that dehydrins play specific
protective roles against water stress in plant cells, by preventing denaturation of macromolecules (Sun et al., 2009) and
safeguarding membrane structure (Mouillon et al., 2008).
Although differential dehydrin expression has been
shown to change in response to drought in several species,
few reports on dehydrin expression in Phaseolus vulgaris L.
are available. Since dehydrin presence has been associated
with stress tolerance, they have been proposed as markers
for selecting drought tolerant plants (López et al., 2002).
This work profiled the presence of dehydrins in leaves and
pods of cv. ‘Otomí’, a Mexican semi-arid highland bean cultivar, classified as water stress-adapted during the vegetative and reproductive stages.
MATERIALS AND METHODS
In Arabidopsis thaliana, overexpression of the wheat dehydrin DHN-5 contributed to enhanced osmotic stress tolerance and preservation of in-vitro enzyme activities, which
protected the plant from adverse effects induced by heating
(Brini et al., 2010). Dehydrin synthesis and accumulation
occur not only as a response to stress, but they also take
part in pre-programmed events during later developmental
stages of seed development in orthodox-type seeds (Close, 1996; Tripepi et al., 2011; Jiménez-Bremont et al., 2012).
At these stages, different molecular weight dehydrins have
been found in seeds of Arabidopsis (Olvera-Carrillo et al.,
2010), barrel clover (Medicago trunculata Gaertn.) (Chatelain et al., 2012), barley (Hordeum vulgare L.), onion
(Allium cepa L.), cotton (Gossypium hirsutum L.), tomato
(Solanum lycopersicum Mill.), radish (Raphanus sativus L.),
cowpea (Vigna unguiculata L.), cucumber (Cucumis sativa
L.), pine nut (Pinus spp.), ginkgo (Ginkgo biloba L.) (Close
et al., 1993), and common bean (Colmenero-Flores et al.,
1999). Additionally, dehydrins are constitutively expressed
in vegetative organs like leaves during normal growth conditions (Rorat et al., 2006).
Plant growth conditions, drought stress treatments
and yield evaluation. Seeds of cv. ‘Otomí’ were sown in 6
L plastic bags filled with a 2:2:1:1 (v/v) mixture of soil:river
sand:peat moss:agrolite. The ‘Otomí’ cultivar was selected
because it has shown good agronomic performance at
semi-arid highlands regions of México (Schneider et al.,
1997). Experiments were setup under greenhouse conditions at Montecillo, Texcoco, State of México, located at 19°
54’ N, 98° 54’ W and 2250 masl. Field capacity (FC) and
permanent wilting point (PWP) for the substrate were
determined via the pressure pot and pressure membrane
protocols (Castellanos et al., 2000; Insunza et al., 2010); a
moisture retention curve was generated with these data
(data not shown).
Irrigated (I) plants were maintained at FC (22.5 % of
moisture content) by daily replenishing the water lost via
evapotranspiration in each pot. Daily water loss was determined by pot weight and the estimated values from the
moisture retention curve. Drought stress treatments started when the substrate reached PWP (11.5 % of moisture
content). Daily irrigation was halted and resumed 10 d later
to restore FC conditions in each pot.
Common bean is the most important source of protein
for direct human consumption in the world, most particularly in Latin America and in Eastern and Southern Africa.
Bean seed is also a source of vitamins, dietary fiber, and minerals, and it is free of unsaturated fatty acids (De la Fuente
et al., 2011). Latin America and Africa contribute nearly 8
million tons to the approximately 12 million metric tons
produced annually worldwide (FAO, 2005).
For dehydrin analyses in leaves, four separate drought
stress treatments were imposed at the beginning of four specific developmental stages: 1) seedling stage (S), plants with
three leaves; 2) flowering stage (F); 3) pod formation (PF),
floral opening (approximately 4 d after flowering); and 4)
seed filling period (SF), 11 d after floral opening (dafo). According to Fernández et al. (1991), the corresponding bean
developmental stages were 1) V4; 2) R6; 3) R7; and 4) R8.
In México, common bean is sown in 1.5 million ha (OEIDRUS, 2011), mainly at the highland Northern plains,
located between 1800 and 2200 masl, under rainfed conditions with less than 450 mm of rain per year (González
and Bernsten, 2005). Nuñez et al. (2005) have reported that
drought stress significantly reduces seed yield (by up to 60
%) in bean production. In developing countries, most of
the common bean is produced under low-input agriculture
For dehydrin analyses in pods, samples from plants under
water stress at PF and SF stages (Figure 1) were taken. Control samples were taken from plants irrigated (I) throughout
the growing season, one for dehydrin expression in leaves
60
CASTAÑEDA-SAUCEDO et al.
Rev. Fitotec. Mex. Vol. 37 (1) 2014
and another for dehydrin expression in pods.
sponding to dehydrin proteins (Close et al., 1993), kindly
provided by Dr. T. J. Close. Reactive bands were detected
with a secondary antibody conjugated horseradish peroxidase (Goat anti-Rabbit IgG [H+L]-HRP conjugated,
ZYMED).
Both irrigated (I) and drought stressed treatments (D)
were distributed under a complete randomized blocks design with three replications. The experimental unit was a
group of 20 pots, each with one plant per pot.
The resulting protein-antibody complex was detected by
a chemiluminescent detection system (ECL, Amersham).
Western blots were repeated at least three times. Band density, corresponding to proteins detected with the anti-dehydrin antibody, was obtained in optical density units (ODU)
per mm2 using the analytical program Quantity One 42.1®
(Bio Rad), and a relative intensity band comparison was
performed. Data were analyzed with the SAS software (version 6.12), through ANOVA and Tukey tests (α = 0.05).
During the growing season, temperature and relative
humidity in the greenhouse varied from 17 to 23 °C and
from 57 to 75 %, respectively. Leaf and pod water potentials
(Yl and Yp) were determined at each stage with a Scholander pump, model A699® (Soil Moisture Equipment Corp.
Santa Barbara, CA, USA). At the end of the season, the following variables were measured: dry seed yield per plant
(g), pods per plant, seeds per plant, and seeds per pod. Data
were analyzed with the Statistical Analysis System software
(SAS, 1989-1996, version 6.12), by ANOVA and multiple
mean comparisons with the Tukey test (α = 0.05).
RESULTS AND DISCUSSION
Water status in leaves and pods. At the end of the
drought stress treatments, the average leaf water potentials
for water stressed plants (Yl) were -1.1 MPa at flowering
(F), -1.1 MPa at pod formation (PF), and -1.2 MPa at seed
filling (SF), compared to -0.64 MPa at the same stages under irrigated conditions (I). Pod water potentials (Yp) were
-1.23 and -1.52 MPa for water stressed pods at PF and SF,
respectively, while in watered conditions it was -0.73 MPa.
According to Guida et al. (2004), a mild drought stress for
common bean corresponds to Yl = -0.9 MPa during the preflowering stage. The stress level achieved in the present study is between moderate and severe.
Collected tissues. Mature leaves were collected from irrigated and non-irrigated plants at 0, 6, and 10 d of water
withholding at the S, F, PF, and SF stages. Pods were collected at 4, 7, and 10 dafo (4, 7, and 10 d without watering)
for the PF stage; and at 14, 18, 22 and 30 dafo (3 and 7 d of
stress, and 1 and 7 d of rehydration after the 10 d period of
water withholding, respectively) for SF stage (Figure 1). All
samples were frozen in liquid nitrogen and stored at -20 °C
until analyzed.
Protein extraction and dehydrin identification by
Western blot. Tissue samples from each tissue, 250 mg
fresh weight, were ground up in a mortar with liquid nitrogen and mixed with 1 mL of extraction buffer (20 mM TrisHCl [pH 7.5], 0.5 M NaCl). The extract was centrifuged at
14 000 Xg for 20 min at 4 °C, and the supernatant collected
into Eppendorf vials. Then vials were boiled in a water bath
for 15 min, placed on ice for 10 min, centrifuged again for
20 min, and the supernatant stored at -20 °C until analysis.
Protein was quantified by the Bradford method (1976), using bovine serum albumin as standard.
Compared to controls, drought stress caused seed yield
losses of 1.2 g (10 %), 7 g (57 %), and 6.1 g per plant (50
%) when the stress was applied at the F, PF and SF stages,
respectively (Table 1). Water stress caused highest yield
losses when applied during pod formation and seed filling.
These results might be a result of drought-related downregulation of several basic biosynthetic functions, including
photosynthesis, photorespiration, and amino acid and carbohydrate metabolism, as reported by Neslihan et al. (2002)
and Cuellar et al. (2008).
For Western blot analysis, samples with 10 µg of total
protein extracted from leaves and pods from I and D plants
were subjected to SDS-PAGE (10 % w/v acrylamide) using
Mini Protean II® cells (Bio-Rad), and then transferred to
nylon membranes (11467-065, Gibco BRL) in the blotting
system Trans-blot SD® (Series No. 221 BR 27584, Bio-Rad)
(Towbin et al., 1979). Membranes were blocked in 5 % (w/v)
non-fat dried milk in TBS-T buffer (20 mM Tris-base [pH
7.6], 137 mM NaCl, 0.1 % Tween 20) during 1 h. The membrane was then incubated overnight with the anti-dehydrin
primary antibody, at a 1:1000 dilution. The antibody was
raised against the conserved consensus, carboxyl-end, oligo-peptide sequence TGEKKGIMDKIKEKLPGQH, corre-
When water was withheld at PF and SF stages, yield losses
were closely associated to reductions in the number of seeds
per plant, pods per plant, and seeds per pod. Similarly, Gebeyehu et al. (2010) reported that pods per plant and seeds
per pod are the most affected yield components in droughttreated bean plants. The smaller yield decrease observed at
water stressed plants at the F stage can be attributed to a
30-day delay in flowering; plants partially compensated the
lower production by growing new pods and seeds after the
stress period.
Dehydrins in leaves. In bean leaves, two dehydrin-like
61
DEHYDRIN PATTERNS IN DROUGHT STRESSED BEAN PLANTS
Rev. Fitotec. Mex. Vol. 37 (1) 2014
Flowering
Sowing
Floral opening
analysis
Leaf
Pod analysis
S
F
PF
SF
0 4
7
10
14
18 22
30 dafo
Drought (10 days)
Irrigation (variable time)
Figure 1. Application timing of the drought treatments imposed to bean plants. The substrate was maintained at field
capacity during the irrigated period. Drought stress started when substrate reached the PWP (11.5 % of moisture
content), and was kept in this condition for 10 days by withholding irrigation (dark bars), at the phenological stages
indicated (S, seedling; F, flowering; PF, pod formation; SF, seed filling). After drought stress, irrigation was resumed.
Leaf tissue samples were taken at 0, 6 and 10 days from plants submitted to the four different drought treatments. Pod
tissue samples were collected at the indicated days after floral opening (dafo) from drought-stressed plants during pod
formation (PF) and seed filling (SF).
Table 1. Seed yield and its components in common bean cv. ‘Otomí’ under irrigation (I), and
drought-stressed at flowering (F), pod formation (PF), and seed filling (SF).
Treatment
Yield (g/plant)
Pods per plant
Seeds per plant
Seeds per pod
I
12.2 a
8.8 b
35.0 a
3.97 a
F
11.0 b
11.1 a
36.4 a
3.27 b
PF
5.2 c
5.3 c
16.2 b
3.07 b
SF
6.1 c
5.9 c
19.2 b
3.30 b
1.114
1.431
5.073
0.294
MSD0.05
Means with the same letter in a column are not statistically different (Tukey, 0.05). MSD = minimum significant difference.
proteins of 82 and 73 kDa were detected by Western blot.
These dehydrins were detected in plants grown under water
stress and control conditions at all analyzed phenological
stages (Figure 2). Therefore, both dehydrins appear to be
constitutive in leaves of the ‘Otomí’ cultivar. This behavior might indicate adaptation to drought, since this cultivar
has been selected for Mexican semi-arid highlands regions
(Martínez et al., 2008). Constitutive expression of dehydrin
DHN1 has been detected in vegetative tissues of Vitis vinifera and wild V. yeshanensis, species that have shown tolerance to drought and cold, as well as moderate resistance to
powdery mildew under normal growth conditions (Yang et
al., 2012).
ed plants (Figure 2). It might be possible that the protective
role of these dehydrins against drought was more needed at
these stages of development when seedlings and seeds are
being formed.
Under irrigation, the levels of the 82 and 73 kDa proteins
in leaves tended to diminish from one developmental stage
to the next one (S to F, F to PF, and PF to SF), although they
also showed some increases in each stage. These patterns
strongly suggest that the expression of these dehydrins respond to developmental cues. In common bean seedlings,
Colmenero-Flores et al. (1999) observed accumulation of
PvLEA-18 gene, of their transcripts and of its 14 kDa LEA
encoded protein, in different organs regardless of irrigation.
The authors suggested that accumulation of PvLEA-18 transcript is probably modulated by an ABA-mediated mechanism during water stress and by other hormone-mediated
mechanism involved in growth induction.
Some differences in the amount of both proteins were detected on the 6th and 10th day of stressed plants in any stage,
but only on the 6th day of the S and PF stages the amount of
these proteins was higher in stressed plants than in irrigat-
62
Rev. Fitotec. Mex. Vol. 37 (1) 2014
D
I
D
I
kDa
82
73
0d
6d
Flowering
D
I
10 d
D
I
D
I
kDa
82
73
0d
6d
10 d
Pod formation
D
I
D
I
D
I
kDa
82
73
0d
6d
10 d
Seed filling
D
I
D
I
D
I
kDa
82
73
0d
6d
10 d
Dehydrin content (ODU)
I
Dehydrin content (ODU)
D
Dehydrin content (ODU)
Seedling stage
Dehydrin content (ODU)
CASTAÑEDA-SAUCEDO et al.
D
I
80
60
40
20
0 6 10
82 kDa
0
6 10
73 kDa
D
I
80
60
40
20
0 6 10
82 kDa
0 6 10
73 kDa
D
I
80
60
40
20
0 6 10
82 kDa
80
0 6 10
73 kDa
D
I
60
40
20
0 6 10
0 6 10
82 kDa
73 kDa
Drought stress (days)
Figure 2. Western blots (left panels) and densitometries (right panels) of dehydrin proteins in leaves of common bean cv.
‘Otomí’ at different drought stress treatments. Proteins were extracted from leaves at 0, 6 and 10 days in drought-stressed
treatments (D) or from irrigated (I) plants. Drought treatments were applied at the developmental stages of seedling, flowering, pod formation and seed filling. Proteins (20 mg per lane) were resolved in a PAGE-SDS gel, blotted onto nylon membranes, and decorated with an antibody against a consensus dehydrin amino acid sequence. Dehydrin content was quantified
by densitometry, and columns graphs constructed. ODU: Optical density units/mm2. Vertical lines on the bars are standard
errors (n = 3).
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DEHYDRIN PATTERNS IN DROUGHT STRESSED BEAN PLANTS
Rev. Fitotec. Mex. Vol. 37 (1) 2014
The levels of two dehydrin polypeptides of 40 and 42 kDa
in protein extracts from Oleae europaea leaves (Tripepi et
al., 2011) and of 31 and 40 kDa in leaves of warm-season
Bermuda grass (Cynodon dactylon L.), increased when the
plants were exposed to water stress (Hu et al., 2010). In
grapevine, dehydrins DHN1, DHN2, DHN3, and DHN4
belong to a dehydrin family that might be constitutively
expressed in vegetative tissues, induced during embryogenesis or by drought and other stresses, depending on the
member; thus, each dehydrin exhibits a very distinctive expression pattern, pointing to versatility in dehydrin responsiveness within a single plant species (Yang et al., 2012).
al., 2002), and 50 kDa protein in Quercus ilex (Turco et al.,
2004). However, the tissue, as well as the extent of water
deficit and the genotypic level of drought tolerance (Hu et
al., 2010), determine type, size, and accumulation level of
dehydrins (Close et al., 1993).
Proteins of 82 and 73 kDa (the same ones detected in
leaves), consistently appeared under all the analyzed conditions, possibly indicating they are constitutive proteins
in leaves and pods (Figure 3). A distinct developmentdependent accumulation of 82 and 73 kDa dehydrins was
observed (Figure 4B, 4C). Drought stress applied at the PF
stage caused accumulation reductions at 4 and 10 d of water
withholding, corresponding to 4 and 10 d after floral opening (dafo). In contrast, a sharp increase in 82 kDa protein
at 7 d of water deprivation (7 dafo) was detected (Figure
4B). Large increments at the beginning of the seed filling
period (14 dafo), followed by decreases and stable levels later on, were observed independently of the water treatment
for both proteins (Figure 4B, 4C).
The levels of the 82 and 73 kDa dehydrin proteins in bean
leaves tended to diminish as water stress advanced, in most
stages studied. Decline in the leaf dehydrins accumulation
of 25 and 40 kDa has also been reported in six varieties
of soybean when grown under drought stress conditions
(Laras et al., 2013). Moreover, other protective proteins synthesized in response to osmotic stress induced by mannitol,
such as heat shock proteins and the dnaK-type molecular
chaperone, also decreased their expression after the first 24
h of mannitol treatment; this behavior suggests that these
proteins may be effective at the beginning of the stress period (Zhang and Comatsu, 2007).
When drought stress was applied at SF stage, it caused a
40 % decrease in 82 kDa protein level after 4 d of treatment
(14 dafo), whereas an important increase of 58 % occurred after 8 d of treatment (18 dafo) (Figure 4B). Also at
this stage, the level of 73 kDa protein diminished around
30 % after 3 d of stress. During recovery of water stresses
imposed at the PF or SF stages both proteins matched the
control levels (Figure 4). A decrease in the content of dehydrins after drought stress is not uncommon. Under severe
stress, degradation of pre-existing dehydrins can take place
in association with increases in free amino acids, as well as
a reduction of total protein synthesis, and ribosomes dissociation (Rodríguez et al., 2003; Saladin et al., 2003).
In general, development and drought stress affected 82
and 73 kDa leaf proteins accumulation similarly (Figure
2). The expression profile of a dehydrin gene family in apple (Malus domestica Borkh.) revealed that nine MdDHN
dehydrins are selectively expressed as groups in different
tissues, under normal growing conditions. Under drought
treatment, most members were up regulated more than
100-fold, whereas the rest were induced less than 10-fold
(Liang et al., 2012). It has been suggested that selective expression of DHN1, DHN2, DHN3, and DHN4 proteins in
grapevine tissues is coordinated by cis-regulatory elements
in the promoter region of their genes (Yang et al., 2012).
Thus, the 82 and 73 kDa dehydrins identified in bean leaves
might be members of a protein family and functionally related to each other, implying that they might have similar
cis-regulatory regions at their genes promoters.
The level of 36 kDa dehydrin was affected by drought
stress applied at the PF stage on the 7th day, causing a 15fold increase over the control (Figure 4H). Consequently,
like in leaves, a protective role of 82 and 36 kDa dehydrins
against drought in pods would be more evident at a specific
developmental stage and time after the water stress period
application: 7 d in pod formation and 8 d in seed filling.
Dehydrins might be functioning as stabilizers and protectants both in leaves and pods of stressed plants (Laras et
al., 2013), by interlinking themselves with intracellular macromolecules and covering them with a layer of cohesive
water preventing protein precipitation (Close, 1996), or by
serving as space fillers to prevent cellular collapse at low
water activities (Tunnacliffe and Wise, 2007; Hanin et al.,
2011). Thus, the accumulation of the 82, 73, and 36 kDa dehydrins might contribute to dehydration tolerance in common bean plants. In Poa bulbosa and apple, dehydrins that
show constitutive accumulation can also be stimulated by
water stress (Volaire et al., 2001; Liang et al., 2012).
Dehydrins in pods. Dehydrin accumulation during
pod development was analyzed in response to drought
stress applied at two different stages, pod formation and
seed filling (Figure 1). Anti-dehydrin antibody revealed
nine protein bands of 158, 82, 73, 63, 54, 46, 41, 36, and
22 kDa (Figure 3), expressed at different times during pod
development. Reports indicate that several dehydrin sizes
are expressed across species grown under drought stress;
for example, 25 and 60 kDa proteins in barley, 40 and 60
kDa proteins in maize (Close et al., 1993), 63 kDa protein
in wheat, rice (Oryza sativa L.), and maize (Borovskii et
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CASTAÑEDA-SAUCEDO et al.
Rev. Fitotec. Mex. Vol. 37 (1) 2014
I
PF
SF
I
PF
SF
kDa
I
PF SF
kDa
158
82
73
82
73
54
46
41
41
36
4d
I
PF
SF
I
7d
PF
SF
kDa
10 d
I
PF
SF
kDa
I
PF
82
73
82
73
SF kDa
82
63
36
22
14 d
18 d
22 d
30 d
Figure 3. Western blots of dehydrin proteins detected in pods of common bean cv. ‘Otomí’ at different drought stress treatments. Proteins were extracted from pods of watered (I) and drought stress plants, at pod formation (PF) and seed filling (SF)
stages. Samples were taken at 4, 7, 10, 14, 18, 22 and 30 days after floral opening (d). Proteins (20 mg per lane) were resolved
in a PAGE-SDS gel, blotted into nylon membranes, and decorated with an antibody against dehydrin consensus sequence.
Both irrigated and water stressed bean plants showed
that their levels of three dehydrins considerably increased
at later stages of pod development, without any difference
between water treatments. For example, the 63 and 36 kDa
dehydrins accumulated at 30 dafo, while the 22 kDa protein
accumulated at 22 dafo (Figure 4D, 4H, 4G). On these dates, regular watering had been resumed, so that the regulation in the expression of these dehydrins would completely
rely on a developmental program and not on drought. The
late expression of these proteins during pod development
suggests that they might be involved in the acquisition of
desiccation tolerance by seeds. The presence of diverse dehydrins at this developmental stage has been widely documented for different plant species (Wechsberg et al., 1994;
Bhattarai and Fettig, 2005; Yang et al., 2012).
Expression of proteins of 158 kDa, 54 kDa, 46 kDa, and
41 kDa in pods of plants submitted to drought stress at SF,
precisely at 10 dafo, was peculiar since plants were not yet at
the drought stress phase (Figure 4A, 4E, 4F, 4G). However,
because the beginning of stress was marked as the day the
substrate reached PWP, 11 dafo or 2 d after irrigation had
been halted, it is possible that plants were already stressed,
previously to the SF period; at early stages of embryogenesis, they synthesized these specific proteins to protect the
embryo against drought stress.
The fact that strong and clear increase in dehydrin
synthesis in response to drought stress in the ‘Otomí’ cultivar was not found does not necessarily mean that the presence of other dehydrins is unrelated to water stress resistance. Analysis of bean cultivars adapted to irrigated fields
65
DEHYDRIN PATTERNS IN DROUGHT STRESSED BEAN PLANTS
120
I
PF
SF
Rev. Fitotec. Mex. Vol. 37 (1) 2014
A) 158 kDa
B) 82 kDa
C) 73 kDa
D) 63 kDa
E) 54 kDa
F) 46 kDa
G) 41 kDa
H) 36 kDa
I) 22 kDa
80
40
Dehydrin protein (ODU)
0
60
40
20
0
60
40
20
0
Dafo 4
7
PF
10
Stress
14
18
22
SF
30
4
7
PF
10
Stress
14 18
SF
22
30
4
7
PF
10
Stress
14
18
22
30
SF
Figure 4. Quantification of dehydrins detected in pods of irrigated and water stressed bean cv. ‘Otomí’ plants. Dehydrin
content in protein extracts of bean pods (Figure 3) from irrigated (I) or drought-stressed plants at pod formation (PF) or
seed filling (SF) stages, was quantified by densitometry, and then graphs were constructed. Each graph represents the data
for one dehydrin identified by its size in kiloDaltons (kDa). ODU: Optical density units/mm2. Vertical line on the bars are
standard errors (n = 3).
is necessary to define if some of the dehydrins present in cv.
‘Otomí’ are the result of genetic selection for arid highlands.
It would then be possible to use them as selection markers
for drought resistance cultivars.
kDa were detected in leaves and pods, throughout all the
studied vegetative and reproductive stages of development,
either with or without water stress. These results suggest
the proteins found are constitutive dehydrins in this cultivar. Expression profiles, done early in water deprivation
treatments, for leaves at seedling and seed filling and pods
at seed filling, indicate that these two dehydrins might play
also a relevant protective role against drought at seedling
development and seed formation. Dehydrins of 63, 36, and
22 kDa were highly expressed at the end of the SF stage in
irrigated and water-stressed plants, probably as means of
acquiring desiccation tolerance in seeds.
CONCLUSIONS
Drought stress applied at stages of pod formation and
seed filling had a stronger effect on seed yield than when
applied in previous stages of bean development. Nine different dehydrins were observed in leaves and pods of
common bean cv. ‘Otomí’. Two dehydrins of 82 and 73
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Rev. Fitotec. Mex. Vol. 37 (1) 2014
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