BRAZILIAN JOURNAL OF PLANT PHYSIOLOGY
             The official journal of the SHORT COMMUNICATION
    Brazilian Society of Plant Physiology
Effect of hydrogen cyanamide on the endogenous
hormonal content of pea seedlings (Pisum sativum L.)
Eric Guevara1, Víctor M. Jiménez1*, Jorge Herrera1 and Fritz Bangerth2
1CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica. 2Institute for Special Crop Cultivation and Crop
Physiology (370),  University of Hohenheim, D 70593, Stuttgart,  Germany. *Corresponding author:
victor.jimenez@ucr.ac.cr
Received: 15 May 2008; Returned for revision: 23 June 2008; Accepted: 01 July 2008
Hydrogen cyanamide (HC) has been used to break bud and seed dormancy and to improve rooting in several species,
responses usually associated with the action of plant hormones. However, very few studies have measured endogenous
hormones after HC treatment. Therefore, pea (Pisum sativum L.) seedlings with two fully developed internodes above
the first leaf were sprayed with 0, 0.1 and 0.3% (v/v) HC. Endogenous concentration of indole-3-acetic acid (IAA),
abscisic acid (ABA), zeatin/zeatin riboside and N6(∆2-isopentenyl) adenine/ N6(∆2-isopentenyl) adenosine were
measured by radioimmunoassay 31 and 80 h after HC treatment. A significant increase in ABA and cytokinin (CK) levels
was observed 31 h after treating the plants with 0.3% HC. Small necrotic spots were also noticed in this treatment, thus
revealing a toxic effect of this treatment. Additionally, at 80 h, a significant increase in IAA was found for both HC
concentrations applied. The action of HC upon ABA, CKs and IAA endogenous levels is discussed.
Key words: abscisic acid, cytokinins, indole-3-acetic acid
Efeito da cianamida hidrogenada sobre o conteúdo de hormônios endógenos em plântulas de ervilha (Pisum sativum L.):
A cianamida hidrogenada (CH) tem sido usada para quebrar a dormência de sementes e estimular o enraizamento em
várias espécies, respostas estas usualmente associadas com a ação de hormônios de plantas. Todavia, em muito poucos
estudos se tem avaliado os níveis endógenos de hormônios após a aplicação de CH. Portanto, plântulas de ervilha
(Pisum sativum L.), com dois internódios completamente desenvolvidos acima da primeira folha, foram pulverizadas com
soluções de CH a 0; 0,1; e 0,3% (v/v). Concentrações endógenas de ácido indol-3-acético (AIA), ácido abscísico (AAB),
zeatina/ribosídeo de zeatina e N6(∆2-isopentenil) adenina/ N6(∆2-isopentenil) adenosina foram medidas por
radioimunoensaio após 31 e 80 h da aplicação de CH. Observaram-se aumentos significativos nas concentrações de
AAB e citocininas 31 h após o tratamento das plantas com 0,3% de CH. Verificaram-se pequenas manchas necróticas nas
plantas desse tratamento, que se revelou, pois, tóxico. Adicionalmente, observou-se um incremento na concentração de
AIA após 80 h da aplicação de CH, tanto a 0,1 como a 0,3%. Discute-se a ação da CH sobre as concentrações endógenas
de AAB, AIA e citocininas.
Palavras-chave: ácido abscísico, ácido indol-3-acético, citocininas
Hydrogen cyanamide (HC) is widely used for breaking cyanamide has also been effective in breaking dormancy in
bud dormancy in several deciduous fruit crops in regions seeds of peanut (Alizaga et al., 1992) and oil palm (Herrera et
with insufficient low temperatures during winter time. This al., 1998), and in potato tubers (Northcott and Nowak, 1988;
compound is commonly applied to fruit species such as Guevara and Herrera, 1989). Other HC responses suggest
grapevine, kiwi, apple, peach and other stone fruits involvement of endogenous plant hormones in the reaction
(Mahhou et al., 2003; Pérez and Lira, 2005). Hydrogen to this compound. For example, the seedlings developed
Braz. J. Plant Physiol., 20(2):159-163, 2008
160 GUEVARA et al.
from oil palm and peanut dormant seeds treated with HC to Ten days later, when at least two fully developed internodes
induce germination show strong root development, as above the first leaf were formed, plants were divided in three
compared to the control treatment and to the application of a homogenous groups, each treated with one of the following
cytokinin (CK) (Alizaga et al., 1992; Herrera et al., 1998). HC concentrations: 0, 0.1 and 0.3% v/v of active ingredient
Moreover, it has been observed that HC stimulates growth (Dormex, 49% HC, SKW, Trostberg, Germany) containing
and development of roots in cuttings of several species one drop of Tween 20 per 100 mL solution. The treatments
(Guevara and Jiménez, unpublished results), which may were sprayed on the aerial part of the plants with a manual
suggest interaction with auxins. sprayer, ensuring complete coverage of the plant surface.
Supporting the hypothesis that HC acts, at least Samples were collected 31 and 80 h after treating the
partially, by inducing changes in concentrations of plants with HC. Each sample comprised the aerial part
endogenous hormones, Northcott and Nowak (1988) (including leaves) of 15 plants (cut above the vermiculite
observed an initial reduction in polyamine concentration in level). Samples were immediately frozen in liquid nitrogen
HC-treated potato tubers, which increased when tubers and stored at –20°C until further processing. Three samples
started to sprout. Additionally, Lombard et al. (2006) were collected for each treatment and sampling date.
measured an increase in the zeatin riboside (ZR) For extraction of IAA, ABA, zeatin (Z) /ZR, and N6(∆2-
concentration after treating grape plants with HC. However, isopentenyl) adenine/ N6(∆2-isopentenyl) adenosine (iP/
a general overview could not be gained from those studies iPA), 1.5 g of the frozen samples were ground in liquid
because only one hormone group was analyzed in each nitrogen and then homogenized with 100 mL 80% (v/v)
case. Very recently, we found that treatment of oil palm methanol with an Ultra Turrax (Janke and Kunkel, Staufen,
seeds with HC mainly caused an increase in the IAA Germany) and incubated overnight at 4°C. Purification and
concentrations in embryos and endosperm during the determination of the above-mentioned hormones were
imbibition phase (Jiménez et al., 2008). In this latter work, performed as described by Jiménez et al. (2005). Briefly, the
ABA, gibberellins GA , GA  and GA , and CKs were also extract was filtered through G4-glassinterfilters (max. pore1 3 20
measured. However, no study has been conducted to size 10-16 µm; Schott, Mainz, Germany), then dried under
simultaneously analyze changes in endogenous levels of low pressure and dissolved in 12 mL 0.1 M ammonium
several plant hormones following HC application directly to acetate (pH 9.0) by using an ultrasonic bath and
plants. Since pea has been used as a “model plant” in many subsequently frozen at  20°C overnight. After thawing, the
studies on hormonal regulation of several physiological extract was centrifuged at 4°C for 25 min at 22000 g to remove
processes, such as apical dominance and leaf development macromolecular substances like lipids, proteins, etc. For
(Demason and Chawla, 2006; Wang et al., 2006; and further purification, the supernatant from the centrifugation
references therein), this species was chosen to conduct the step was passed through a preconditioned column
experiment described below. combination, assembled in the following sequence: PVP
Pea seeds (Pisum sativum L. cv. Lisa) were surface- (Sigma, Deisenhofen, Germany); DEAE Sephadex A-25
sterilized with sodium hypochlorite (1%, w/v) for 5 min and (Pharmacia, Freiburg, Germany); C18 Sep-Pak cartridge
then washed three times with distilled water. They were (Waters, Eschborn, Germany). The extracts were first loaded
subsequently imbibed in small trays containing enough onto the syringe filled with PVP and eluted with 30 mL 0.01
water to slightly cover the seeds. After 2 d, seeds with a M ammonium acetate (pH 7.5). At this point, the acidic
visible radicle were planted in trays containing wet hormones (IAA, ABA) were bound as anions to the DEAE
vermiculite and grown at 25°C with a 16 h photoperiod Sephadex, while the CKs passed through to the Sep-Pak
provided by 400 W HQI lamps (Osram, Munich, Germany), cartridge. This cartridge was then replaced by a second one
which gave a photon flux density of approximately 500 µmol preconditioned for ABA. The PVP column retaining the
m-2 s-1. The level of water was checked daily and the plants phenolic substances and other impurities was discarded,
were watered when necessary. Eight days later, when the and ABA eluted from the DEAE Sephadex into the second
first unifoliate leaf above the cotyledons was visible, plants cartridge with 15 mL 0.75 M ammonium acetate. Finally the
were fertilized at the base of 2 mL L-1 of Wuxal complete second cartridge was replaced by a third one
nutrient solution (Aglukon GmbH, Düsseldorf, Germany). preconditioned for IAA, which was then eluted from the
Braz. J. Plant Physiol., 20(2):159-163, 2008
EFFECT OF HYDROGEN CYANAMIDE ON THE ENDOGENOUS HORMONAL CONTENT OF PEA SEEDLINGS 161
Sephadex into this cartridge with 10 mL 2.0 M ammonium reactive oxygen species (ROS), such as H2O2, may cause
acetate. Thereafter the cartridge containing the CKs was lipid oxidation and, as a result, necrosis of the tissues
washed with 4 mL 0.01 M ammonium acetate and the (Apel and Hirt, 2004). Although a direct toxic effect of
cartridges with the acidic hormones with 4 mL 0.1 M acetic Tween 80, a related surfactant to the one used in this
acid. All hormones were then eluted from the cartridges with experiment,  has been reported in other species
4 mL of the following solutions: 30% methanol for Z/ZR; (Thimmaraju et al., 2003), the absence of damage at the
80% methanol for iP/iPA; 40% methanol in 0.1 M acetic acid lower HC concentration and control plants, points to
for IAA and 65% methanol in 0.1 M acetic acid for ABA. toxicity by HC instead.
Using this procedure, inactive and conjugated forms of Indole-3-acetic acid was the only hormone whose
IAA, ABA and CKs were removed from the assay and, endogenous concentration responded to lower HC
therefore, did not interfere with the measurement of the concentration (Figure 1A). No significant differences
active and free forms in the following step. Hormones were between treatments were detected in the IAA levels 31 h
quantified by radio-immunoassay using polyclonal after HC application. However, 80 h after treating the
antibodies. plants, the IAA levels measured in control plants
Overall recovery during the described purification decreased significantly,  while those in both HC
procedures was determined in parallel samples after treatments increased. This may indicate that IAA,
overnight extraction with radiolabelled internal possibly together with H2O2, was among the initial
standards, and ranged between 40 and 70% for IAA, over reactions of the plant to the HC treatment, even at low
90% for ABA and close to 80% for both CKs. Due to the concentrations of this product. This increase in IAA
high variation found in IAA recovery values, an internal could be a response to stress, such as the one mentioned
IAA standard of 1[14C]-IAA (specific activity 518 GBq above, as pointed out by Quirino et al. (1999) for
mol-1; Amersham, Braunschweig, Germany) was included Arabidopsis thaliana and by Havlová et al. (2008) in
in each one of the samples, and the IAA concentration tobacco. This IAA upsurge might trigger other hormonal
results were adjusted accordingly. The other hormones changes. For example, it has been recently demonstrated
were not corrected for losses. by kinetic and molecular studies that auxin herbicides
Endogenous hormonal levels were analyzed using three and IAA by itself can initiate a “reaction chain” that
biological replications and statistical analysis was carried starts with an increase in IAA, or related auxins, which
out using Infostat (Universidad Nacional de Córdoba, induces ethylene biosynthesis, which in turn accelerates
Argentina). The LSD-Fisher Test was used to determine ABA biosynthesis and possibly jasmonic acid
significant differences between means (P < 0.05). production (Grossman, 2007; and references therein).
Thirty-one hours after applying the spray treatments, At 0.3% concentration, HC caused a significant
small chlorotic spots (2-3 mm in diameter) were observed increase in endogenous ABA already after 31 h. Those
on the leaves of plants treated with the higher (0.3%) HC levels were approximately three times higher in this
concentration tested. These spots turned dark brown and treatment than in the control and the lower HC
later became necrotic. This suggests toxic effects of HC concentration (Figure 1B). Increased ABA concentration
at that concentration. Leaf and bud necrosis has already was also maintained after 80 h. The rise in ABA as a result
been reported in vegetative aerial parts of Psidium of the higher HC dose may be more a consequence of
guajava (Quijada et al., 1999) and Citrus latifolia stress [as reviewed by Netting (2000)] than a direct
(Cañizares and Rojas, 2001) plants sprayed with 0.5% HC. stimulation of ABA synthesis by HC. In the latter case an
Actively-growing leaves are, therefore, presumably more increase in ABA should have also been observed with the
sensitive than buds and seeds to low concentrations of lower HC concentration.
HC, a product that is normally used in doses of 2% or Both CKs evaluated behaved similarly to each other.
above for dormant tissues. Pérez and Lira (2005) observed Thirty-one h after treatment, CK concentrations were
a reduced catalase activity in dormant buds of grapevine twice as high in 0.3% HC-treated plants compared to 0.1%
treated with HC, followed by an increase in the H2O2 HC and control. After 80 h, the plants treated with the
levels in the buds. It is known that accumulation of lower HC concentration also showed an increase in CKs
Braz. J. Plant Physiol., 20(2):159-163, 2008
162 GUEVARA et al.
40 40
A c B
d
35 35 c
bc cdbc
30 30
ab
a
25 25
20 20
b
15 15 ab ab
a
10 10
5 5
0 0
2.5 14
C D
0 0.1% 0.3% d
d 12
2.0
c
c 10
1.5 b 8 b
a a
6 a1.0
a
a
a 4
0.5
2
0.0 0
31 h 80 h 31 h 80 h
Time (h) Time (h)
Figure 1. Endogenous levels of indole-3-acetic acid, IAA (A), abscisic acid, ABA (B), zeatin/zeatin riboside, Z/ZR (C)
and N6(∆2-isopentenyl) adenine/ N6(∆2-isopentenyl) adenosine, iP/iPA (D) in pea seedlings 31 and 80 h after hydrogen
cyanamide treatment (0, 0.1%, and 0.3%). Significant differences within the same hormone (P < 0.05) are indicated with
different letters. n = 3 ± SD.
(Figure 1C,D). In control plants the levels of both CKs (2005) found a strong increase in endogenous levels of
remained low without much variation in both evaluations. CKs in French bean and tobacco plants treated with 100
Mild stress, such as that caused by the highest HC µM ABA and afterwards exposed to severe water stress.
concentration in pea, has been associated with The results of this study complement findings of
stimulation of metabolic activity in plants, in an effort to previous investigations in which the mode of action of HC
overcome the adverse conditions. This increase in CK during dormancy breakage in vegetative tissues and seeds
levels could be the consequence of this increased was studied (Northcott and Nowak, 1988; Pérez and Lira,
metabolic activity of stressed plants. Pospíšilová et al. 2005; Lombard et al., 2006; Jiménez et al., 2008). In addition to
Braz. J. Plant Physiol., 20(2):159-163, 2008
-1 -1
Z/ZR (ng g DW) IAA (ng g DW)
-1 -1
iP/iPA (ng g DW) ABA (ng g DW)
EFFECT OF HYDROGEN CYANAMIDE ON THE ENDOGENOUS HORMONAL CONTENT OF PEA SEEDLINGS 163
the participation of catalase and H2O2 and, in consequence, Evolution of endogenous hormone concentration in
of ABA, we observed an increase in endogenous levels of embryogenic cultures of carrot during early expression
IAA and CKs as an effect of HC application. Similar results of somatic embryogenesis. Plant Cell Rep. 23:567-572.
have been observed in oil palm seeds by Jiménez et al. Jiménez VM, Guevara E, Herrera J, Alizaga R, Bangerth F
(2008). These results may suggest that HC or the stress (2008) Changes in hormone concentration during
reaction thereafter affect synthesis or metabolism of IAA dormancy release of oil palm (Elaeis guineensis)
and CKs in plants. However, the reason and function for seeds. Seed Sci. Technol. in press.
these events needs to be investigated. Lombard PJ, Cook NC, Bellstedt DU (2006) Endogenous
Acknowledgments: The authors would like to thank the cytokinin levels of table grape vines during spring
German Academic Exchange Service (DAAD) for financial budburst as influenced by hydrogen cyanamide
support in the form of short-time fellowships to EG and application and pruning. Sci. Hort. 109:92-96.
Mahhou A, Alahoui H, Jadari R (2003) Effets de la
JH and a long-term scholarship to VMJ. Authors also
cyanamide hydrogène et de l’acide gibbérellique sur la
wish to thank anonymous reviewers for helpful
levée de dormance du pommier ‘Dorsett Golden’ au
comments. sud du Maroc. Fruits 58:229-238.
Netting AG (2000) pH, abscisic acid and the integration of
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