Botanical Studies (2008) 49: 343-350.
*
Corresponding author: E-mail: drawahid2001@yahoo.com.
INTRODUCTION
Greater and better synchronized germination is
crucial for achieving an optimal crop stand and better
productivity, but several environmental constraints are
great impediments. One pragmatic approach to increase
crop production is seed invigoration (Lee and Kim, 2000,
Basra et al., 2004; Farooq et al., 2006). Seed invigoration
strategies include hydropriming, osmoconditioning,
osmohardening, hardening, hormonal-priming,
matripriming, and others (Chiu et al., 2002; Kao et al.,
2005; Windauer et al., 2007). The invigoration persists
under adverse field conditions like salinity (Wahid, 2004;
Ahmad et al., 2005; Abdul Jaleel et al., 2007; Wahid et al.,
2007), temperature extremes (Pill and Finch-Savage, 1988;
Bradford et al., 1990, Wahid and Shabbir, 2005), hypoxia
(Ruan et al., 2002), and drought (Du and Tuong, 2002).
Seed priming, accomplished through different means
and methods, enhances pre- and post-germination
activities. Hydroprimed maize seeds showed rapid
seedling emergence and improved field stand (Nagar et
al., 1998), and osmoprimed seeds with PEG, K
2
HPO
4
or
KNO
3
showed accelerated germination (Basra et al., 1989).
Nerson and Govers (1986) found that 2-3% solutions
of KH
2
PO
4
+ KNO
3
(1:1) synchronized and increased
germination rate in muskmelon seeds. Sunflower seeds
treated with PEG-8000 solution at 15¢XC had an increased
germination rate (Bailly et al., 1998, 2000). The use of
plant growth regulators during pre-soaking, priming, and
other seed pretreatments improved crop performance
(Miyoshi and Sato, 1997; Basra et al., 2006). GA
3
and
ethylene stimulated the elongation of embryonic tissues
and internodes of rice seedlings while ABA promoted
mesocotyl elongation (Lee et al., 1999). Dry heat treatment
broke seed dormancy to ensure better seed germination
(Dadlani and Seshu, 1990). Incubation of cotton seed at
60¢XC markedly improved seedling emergence and vigor
(Basra et al., 2004).
The synchronization and promotion of germination
with seed priming may take place for several reasons, but
changes in metabolite levels are important events during
Priming-induced metabolic changes in sunflower
(Helianthus annuus) achenes improve germination and
seedling growth
Abdul WAHID
1,
*, Asma NOREEN
1
, Shahzad M.A. BASRA
2
, Sadia GELANI
1
, and M. FAROOQ
3
1
Department of Botany, University of Agriculture, Faisalabad-38040, Pakistan
2
Department of Crop Physiology, University of Agriculture, Faisalabad-38040, Pakistan
3
Department of Agronomy, University of Agriculture, Faisalabad-38040, Pakistan
(Received July 30, 2007; Accepted May 22, 2008)
ABSTRACT.
Seed priming improves vigor, but priming agents may differ greatly in their effectiveness. The
present study was performed to unravel the physiological basis of vigor improvement by priming sunflower
achenes with pre-optimized levels of hydrogen peroxide (H
2
O
2
), salicylic acid (SA), thiourea (TU), gibberellic
acid (GA
3
), ascorbic acid (AA), sodium chloride (NaCl), freezing and heating. Most of the treatments induced
de novo synthesis of peptides with low (37 kDa for H
2
O
2
, SA and NaCl treatments, and 57 kDa for SA and
TU treatments) and high (157 kDa for H
2
O
2
, SA, TU, GA
3
and AA treatments and 167 kDa for SA treatment)
molecular mass, reduced solute leakage, and an enhanced soluble sugar pool in the achenes. Priming reduced
days to 50% germination (T
50
) and mean germination time (MGT) and improved germination energy (GE) and
final germination percentage (FGP). Shoot length was improved by priming with H
2
O
2
, GA
3
, and NaCl; root
length with NaCl and H
2
O
2
; shoot and root dry weight with H
2
O
2
, SA and AA. Positive correlations between
GE and FGP and expressed peptides, soluble sugars, shoot and root length, and dry weight and negative
ones with EC of leachate suggested that pre-germination changes in primed achenes, in addition to improve
germination, show lasting effects in promoting seedling growth. Of the treatments, H
2
O
2
, SA, TU and GA
3
were the most effective. Overall, the effects of priming treatments are related to de novo protein synthesis, an
improved repair mechanism, and germination substrates for vigorous and earlier production of seedlings.
Keywords: Leachate; Protein synthesis; Seedling vigor; Signaling; Sunflower.
PhySIOlOgy
pg_0002
344
Botanical Studies, Vol. 49, 2008
seed priming. As revealed from microarray studies, seed
protein synthesis is a global phenomenon that initiates
the up- or down-regulation of a number of germination
related genes (Gallardo et al., 2001; Soeda et al., 2005).
Natural or artificial seed priming induces the mobilization
and solublization of globulins and the synthesis of late
embryogenesis abundant proteins (Capron et al., 2000;
Gamboa-deBuen et al., 2006). Antioxidant enzymes¡X
including superoxide dismutase, catalase, and glutathione
reductase¡Xwere also expressed during seed priming
(Bailly et al., 2000). Among other pre-germination
metabolic changes, seed priming decreased the level of
malondialdehyde (Bailly et al., 1998, 2000), changed
saturated and unsaturated fatty acids (Walters et al., 2005),
and induced £\-amylase to increase the soluble sugar pool,
thereby enhancing seedling emergence and other related
attributes (Mwale et al., 2003; Farooq et al., 2006).
The achene of sunflower (Helianthus annuus L.) is
an important source of edible oil. However, sunflower is
an oilseed crop and its germination is very susceptible
to changing field conditions. Its productivity has not
improved appreciably over the past eight years in Pakistan,
despite the introduction of improved germplasm, and this
is largely due to hampered seed/seedling vigor under field
conditions (Anonymous, 2005). In this respect, the use of
seeds with enhanced vigor can be a practicable strategy
to obtain healthy seedlings and a better crop stand under
a range of environmental conditions. Reports showing
priming-induced vigor enhancement are numerous, but
those highlighting the physiological and biochemical basis
of such changes are scarce. We hypothesize that metabolic
changes and effects produced by seed priming treatments
are specific to each treatment, and may be related to a
signaling action. In view of this hypothesis, we evaluated
the physiological basis of pre- and post-germination
changes produced by the most effective priming agent
levels (in terms of improved vigor in sunflower achenes)
and their relationships to germination and seedling growth.
MATERIAlS AND METhODS
Plant material and treatment selection
Achenes of sunflower (Helianthus annuus L. cv.
Hyson-33) were obtained from Oilseed Research Institute,
Faisalabad. For the selection of effective priming
treatments, the achenes were pretreated with varying
levels of hydrogen peroxide (H
2
O
2
), salicylic acid (SA),
thiourea (TU), gibberellic acid (GA
3
), ascorbic acid (AA),
sodium chloride (NaCl), freezing and heating, followed
by drying at 27¢XC to the original (8-10%) moisture. The
most effective levels of these treatments, based on greatest
final germination percentage (FGP), selected for this study
were: H
2
O
2
(100 £gM for 8 h), SA (50 mg L
-1
for 8 h), TU
(10 mg L
-1
for 8 h), GA
3
(150 mg L
-1
for 8 h) AA (500 mg
L
-1
for 8 h), NaCl (1000 mg L
-1
for 8 h), heating (40¢XC for
48 h), and freezing (-19¢XC for 24 h).
Priming-induced metabolic changes in achenes
For protein determinations, frozen seed material (0.5
g) was ground in a pre-chilled pestle-mortar in phosphate
buffer saline (PBS) containing NaCl (137 mM), KCl (2.7
mM), Na
2
HPO
4
(2 mM), and cocktail protease inhibitors (1
mM). The pH was adjusted to 7.2 (Sambrook and Russell,
2001). The amount of total proteins was determined using
a Bradford assay (Bradford, 1976). For fractionation of
proteins, 20 £gg of each sample was treated with lysis-
buffer (sodium dodesyl sulphate 10%, Tris 0.5 M with
pH 6.8, glycerol 80% and bromophenol blue), loaded on
a 12.5% polyacrylamide gel, and electrophoresed at 100
V (Laemli, 1970). The gel was stained in a 0.1% solution
of 20% methanolic Coomassie brilliant blue G-250
overnight. After destaining for 20 min in 20% methanol,
the gel was wrapped in a permeable membrane, dried in
the dark for three days, and scanned, and molecular mass
of the expressed peptides was ascertained by comparing
them with protein markers, which were run alongside the
unknown samples.
For the assessment of solute leakage, the achenes were
put on a double layer of filter paper (Whatman No. 1) in
pairs of petri dishes (9 cm diameter) containing 10 mL of
deionized water and kept overnight at 25¢XC. The leachate
was collected after washing the filter papers. The final
volume was made up to 10 mL, and the sample¡¦s electrical
conductivity (EC) was determined. To estimate soluble
sugar contents, 1 g of the sample was mixed with 10 mL
distilled water and left for 24 h at 25¢XC (Lee and Kim,
2000). The mixture was filtered through Whatman No.
42 filter paper, and the final volume was made up to 10
mL with distilled water. Total soluble sugar contents were
estimated by anthrone regent (Yoshida et al., 1976).
Achene germination
Achenes were sown in petri dishes on a double layer
of moist filter paper and kept at 26¡Ó1¢XC in a plant growth
chamber (Eyelatron, FLI-301N, Tokyo Rikakikai Co.,
Ltd., Tokyo, Japan). An achene was considered germinated
when radicle was 5 mm long. Counts of germinating
seeds were taken daily up to six days after the start of
germination. Time to 50% germination (T
50
) and mean-
germination time (MGT) were calculated according to
Coolbear et al. (1984) and Ellis and Roberts (1981),
respectively. Energy of germination (GE), determined
on the experiment¡¦s 4th day, represented the ratio of
germinated seeds to total seeds. Final germination
percentage (FGP) was determined at the end of
experiment (AOSA, 1983).
Seedling growth
The primed achenes were sown in 2 kg capacity
pots containing sand, which was washed thrice with
distilled water. The seedling growth was supported by
supplementing half strength Hoagland nutrient solution
(Epstein and Bloom, 2005) twice during whole of the
growth period. Fifteen days after sowing, the seedlings
pg_0003
WAHID et al. ¡X Priming effect on sunflower achene germination and growth
345
were harvested. Length and fresh weight (FW) of shoot
and root were determined immediately after harvesting
while dry weight (DW) was determined after drying these
tissues at 80¢XC in an oven for a week.
Statistical analysis
All the experiments were conducted in a completely
randomized design with three replications. The data
recorded for various attributes was subjected to statistical
analysis using COSTAT computer software (COHORT
software, 2003, Monterey, California). For significant
ANOVA tests (P < 0.01), Duncan¡¦s new Multiple Range
Test was applied for the comparison of means, which
were indicated by alphabets on data sets. Correlations
of germination attributes were established with achene
metabolic changes (number of peptides expressed, EC of
leachate and soluble sugars) and seedling growth (shoot
and root length and dry weight) attributes.
RESUlTS
Priming-induced metabolic changes in achenes
Fractionation of proteins indicated the appearance
of low and high molecular mass peptides by priming
treatments, except for control, freezing and heat. Among
peptides of low molecular mass, a 37 kDa peptide was
induced by H
2
O
2
, SA and NaCl, and another 57 kDa
peptide was evident in SA and TU treatments. A high
molecular mass peptide, 157 kDa, was induced by H
2
O
2
,
SA, TU, GA and AA priming while a 165 kDa peptide was
induced by SA (Figure 1). The data indicated significant
differences (P . 0.01) among the priming treatments for
the EC of leachate and soluble sugar concentrations. EC
of achene leachate was highest in control and freezing,
but the lowest in SA and H
2
O
2
. On the other hand, soluble
sugars were greatest in the SA and H
2
O
2
treatments (Figure
1).
Data on germination attributes of achenes revealed
significant (P.0.01) differences among priming treatments
(Table 1). Except for freezing and heating, although all
treatments were effective in curtailing the T
50
, H
2
O
2
and
TU were the most effective. MGT was highest in control
and freezing treatments, but it was lowest in SA and TU
(Table 1). Achene priming greatly improved GE (potential
of achene to germinate vigorously) as compared to control,
being greatest in SA, followed by H
2
O
2
, TU and GA.
Likewise, FGP was improved by all the priming treatments
but most of all by SA, H
2
O
2
and TU (Table 1).
Seedling growth attributes
Shoot and root length, which showed significant (P.
0.01) difference among treatments, was promoted by
all the priming treatments. Increase in shoot length was
greatest in GA
3
, H
2
O
2
, and NaCl primed achenes. Root
length was greatly improved with NaCl priming, followed
by H
2
O
2
and SA, while freezing was at the bottom (Figure
2). All the priming treatments, with significant (P.0.01)
differences, improved shoot and root dry weight, but H
2
O
2
and SA were the most effective in this regard (Figure 2).
Correlations
Parallels were drawn between germination attributes
and priming-induced changes in the achenes and seedling
growth attributes (Table 2). T
50
and MGT showed no
association while GE and FGP were positively related to
Figure 1. Pa tte rn a nd mol ecul ar weight of t he expre ss ed
peptides, electrical conductivity of leachates and soluble sugars
concentrations of sunflower achenes primed with most effective
levels of priming treatments. Bars with same alphabets were not
significantly different (P.0.05).
pg_0004
346
Botanical Studies, Vol. 49, 2008
peptide expression. EC of leachate was positively related
to T
50
and MGT but negatively to GE and FGP. However,
soluble sugars were negatively correlated to T
50
and MGT
but positively to GE and FGP. Shoot and root length and
dry weights were negatively correlated with T
50
and MGT
but positively with GE and FGP (Table 1).
DISCUSSION
This study was performed to determine the varied
responses of germination and seedling growth attributes to
priming-induced metabolic changes in sunflower achenes
in order to obtain a better crop stand in the field. Priming
of seed is an effective tool in enhancing the emergence
and vigor of seedlings under both optimal (Demir and
van de Venter, 1999; Farooq et al., 2006) and suboptimal
conditions (Wahid and Shabbir, 2005; Wahid et al., 2007).
Nevertheless, such effects are linked to priming-induced
metabolic changes. This study, involving eight different
treatments, revealed that sunflower achene priming led
to differential expression of certain low (37 and 57 kDa)
and high (157 and 165 kDa) molecular weight peptides
together with reduced ion-leakage and an enhanced soluble
sugars pool (Figure 1). The H
2
O
2
, SA and TU treatments
produced these changes most effectively.
Although all priming strategies curtailed T
50
and MGT
and enhanced GE and FGP; SA, H
2
O
2
and TU were the
most effective (Table 1), leading to an earlier and energetic
seedling start. Primed achenes evaluated for seedling
production revealed substantial improvement in elongation
and dry mass of both shoot and root (Figure 2), as reported
in earlier studies (McDonald, 2000; Mwale et al., 2003;
Table 1. Some germination attributes of sunflower achenes at the most effective levels of priming treatments
Treatment
Days to 50%
germination
Mean germination time
(days)
Energy of germination
(%)
Final germination
percentage
Control
4.80¡Ó0.44a
8.43¡Ó0.15a
45.67¡Ó4.04d
55.67¡Ó5.03d
Hydrogen peroxide
2.27¡Ó0.17d
6.33¡Ó0.12cd
85.33¡Ó2.31ab
90.67¡Ó2.31ab
Salicylic acid
2.64¡Ó0.27d
6.13¡Ó0.23d
89.33¡Ó6.11a
95.67¡Ó2.31a
Thiourea
2.39¡Ó0.20d
6.37¡Ó0.12d
82.67¡Ó8.08ab
89.33¡Ó7.37ab
Gibberellic acid
2.41¡Ó0.10d
6.40¡Ó0.10cd
82.00¡Ó8.66ab
86.00¡Ó6.93b
Ascorbic acid
2.42¡Ó0.21d
6.47¡Ó0.21cd
80.00¡Ó6.93ab
85.33¡Ó2.31b
Sodium chloride
2.48¡Ó0.15d
6.50¡Ó0.44cd
78.33¡Ó4.61b
83.33¡Ó5.03b
Freezing
3.93¡Ó0.31b
7.20¡Ó0.10ab
58.00¡Ó4.00c
66.00¡Ó4.00c
Heating
3.17¡Ó0.25c
6.83¡Ó0.38bc
62.33¡Ó4.16c
68.67¡Ó2.31c
Means sharing same alphabet were not significantly different (P.0.05).
Table 2. Relationships of germination attributes of sunflower achenes with priming-induced changes in the achenes and seedling
growth attributes
Characteristics
Days to 50% germination Mean germination time Energy of germination Final germination
percentage
Pre-germination changes
No. of peptides
-0.573ns
-0.658ns
0.795*
0.834**
EC of leachate
0.889**
0.923**
-0.930**
-0.936**
Soluble sugars
-0.701*
-0.742*
0.837**
0.866**
Seedling characteristics
Shoot length
-0.840**
-0.782*
0.806*
0.768*
Root length
-0.833**
-0.765*
0.777*
0.747*
Shoot dry weight
-0.670*
-0.727*
0.787*
0.805**
Root dry weight
-0.696*
-0.741*
0.759*
0.767*
Significant at ** P.0.01; * P.0.05; ns non-significant.
pg_0005
WAHID et al. ¡X Priming effect on sunflower achene germination and growth
347
Farooq et al., 2006). For seedling elongation, H
2
O
2
, NaCl
and GA
3
were the best while for shoot and root dry weight
SA and H
2
O
2
were promising. A greater improvement
in elongation and dry weight of shoot than root (Figure
2) revealed that changes in primed achenes diverted a
greater part of the cotyledonary resources towards the
shoot, which was crucial to its earlier establishment and
photosynthesis for vigorous growth. The causes of above
effects produced by achene priming treatments may be
different. Since H
2
O
2
, SA, TU and GA
3
are signaling
molecules (Taiz and Zeiger, 2006), it is plausible that they
reprogrammed the gene expression (Cruz-Garcia et al.,
2003; Soeda et al., 2005; Gamboa-deBuen et al., 2006),
leading to de novo protein synthesis, a membrane repair
mechanism, and more storage proteins and other substrates
available for improved and synchronized germination
(Table 1).
As evident from the above, some priming treatments
were more effective in improving achene germination
attributes than others, a fact which is possibly associated
with pre-germination metabolic changes in the achenes
and post-germination seedling performance. Data showed
positive correlations between the expressed peptides
and soluble sugars with GE and FGP, and negative
ones between soluble sugars and T
50
and MGT. EC of
leachate was positively correlated with T
50
and MGT and
negatively with GE and FGP (Table 2). This revealed
that improvement in germination was closely associated
with de novo protein expression, repair mechanisms, and
a greater availability of germination substrates (Table 1),
which resulted in a rapid and energetic start (Mwale et al.,
2003; Wahid et al., 2007). Likewise, a stronger negative
correlations between T
50
and MGT and a positive one
between GE and FGP separately for shoot and root length
and dry weight further substantiated that the priming-
induced metabolic changes in the achenes had lasting
influence on the seedling growth (Farooq et al., 2006;
Wahid et al., 2007). Thus, curtailed time to emergence
and vigorous seedling start are beneficial effects of seed
priming.
It is concluded that priming-induced improvements in
achene germination and seedling growth were associated
with proteins synthesis, membrane repair mechanisms,
and greater substrate availability for germination. From
the metabolic changes in achenes during priming, it is
plausible that the priming treatments reprogrammed the
gene expression for antioxidant synthesis and mobilized
germination substrates in greater amounts. Among the
treatments, SA and H
2
O
2
, probably using a common
signaling mechanism, proved to be the most effective. Our
results suggest that these strategies can be of great help
in the production of sunflower seed capable of growing
vigorously under contrasting field conditions.
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