Acta Physiol Plant (2017) 39:202
DOI 10.1007/s11738-017-2500-z
ORIGINAL ARTICLE
Polyamines in yellow lupin (Lupinus luteus L.) tolerance to soil
drought
Katarzyna Juzoń1 • Ilona Czyczyło-Mysza1 • Izabela Marcińska1 • Michał Dziurka1
Piotr Waligórski1 • Edyta Skrzypek1
•
Received: 14 November 2016 / Revised: 31 July 2017 / Accepted: 17 August 2017 / Published online: 23 August 2017
Ó The Author(s) 2017. This article is an open access publication
Abstract Polyamines (PAs) are related to many physiological processes, including soil drought stress. Two yellow
lupin ‘Morocco 4’ (drought tolerant) and ‘Taper’ (drought
sensitive) were exposed to soil drought for 2 weeks. The
half of the examined plants were additionally sprayed with
a solution of polyamine biosynthesis inhibitor—DL-a-difluoromethylarginine (DFMA). Yellow lupin leaves
showed a 19% increase and seeds a 54% decrease in the
total PA contents. The seeds contained fourfold less PAs
than the leaves under drought conditions. The highest
amount of spermidine and lack of agmatine were found in
the leaves, while in the seeds the highest content of spermine and the presence of agmatine was confirmed. The use
of DFMA under drought conditions decreased the content
of spermine in ‘Morocco 4’ and ‘Taper’ (41 and 19%,
respectively) and spermidine in ‘Taper’ (by 13%), as well
as reduced two out of three of the yield components. More
tolerant ‘Morocco 4’, after DFMA treatment was characterized by a higher spermidine and spermine content and a
smaller decrease in yield components compared to the less
tolerant ‘Taper’. Simultaneously subjecting plants to soil
drought and DFMA treatment caused in ‘Morocco 4’ a
decline in the number of pods and seeds per plant and seeds
dry weight per plant (64, 50 and 54%, respectively), while
in ‘Taper’ a reduction of the number of pods per plant and
seeds per pod (32 and 27%, respectively) was observed.
These results confirm that PAs are involved in yellow lupin
Communicated by K Apostol.
& Edyta Skrzypek
e.skrzypek@ifr-pan.edu.pl
1
The F. Górski Institute of Plant Physiology Polish Academy
of Sciences, Niezapominajek 21, 30-239 Kraków, Poland
tolerance and may play a protective function under soil
drought conditions.
Keywords Polyamines Soil drought Lupinus luteus L.
DL-a-Difluoromethylarginine
Introduction
Droughts are the most complex phenomena and their
causes and effects are still not well known and understood.
In the last 30 years, droughts have occurred more often
throughout the world, causing great social and economic
damages. Droughts are becoming increasingly frequent;
they are more intense and cover larger areas. Soil drought
seriously limits plant growth, which leads to a significant
decrease in crop yield. It can reduce crop yield even up to
50%. The availability of water is in fact one of the most
important factors increasing plant yield.
A group of plants particularly sensitive to adverse
weather conditions are legume plants, including yellow
lupin. Legumes are known for their characteristic flower
structure and ability to interact with rhizobia as well as
their importance to humans (Graham and Vance 2003). For
decades, legumes have been used in animal nutrition,
mainly to increase the protein concentration in the fodder.
Legume plants contain approximately 30% of protein, with
the most valuable amino acids, essential for the proper
functioning of animal organisms. Therefore, they constitute
a valuable source of protein in the human diet. Unfortunately, improvement in legume crop yields is inferior to
those of cereals. The reasons are unfavorable environmental conditions under which many legume species are
grown. Furthermore, the problem of water shortages for
legumes is getting worse along with the increasing number
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of areas affected by drought (Postel 2000). For legumes,
the most dangerous are water shortages in critical phases,
which are periods of the greatest biomass increase as well
as in the phase of generative organ formation. In legumes,
drought reduces yield by premature and insufficient seed
filling, and as a result of flower and young pod rejections.
There is a vital need to increase drought tolerance in
legumes (Graham and Vance 2003).
Polyamines (PAs) are low molecular organic cations
occurring in a wide range of animal and plant organisms
(Hussain et al. 2011). With respect to plant growth and
development, PAs are widely involved in cell division and
differentiation, morphogenesis, secondary metabolism,
senescence, apoptosis, DNA synthesis, gene transcription,
protein translation and chromatin organization (Yang et al.
2007; Alet et al. 2012; Tavladoraki et al. 2012). In recent
years, the role of PAs in plants against abiotic and biotic
stresses has been thoroughly investigated (Liu et al. 2006).
A generally observed fact is that PA concentration change
under various types of environmental stresses including
drought stress. Stress-tolerant plants are characterized by a
high capacity of increasing PA biosynthesis in response to
stress that can be observed as a two- to threefold increase
of endogenous PA levels compared to unstressed plants
(Kasukabe et al. 2004). PAs play important role in the
stabilization of biologically active polyanionic compounds,
such as cytoplasmic membrane phospholipids, proteins
(including enzymes) or nucleic acids. These compounds
change their activity by interactions with amino groups and
other molecules (Hura et al. 2015). Due to their polycationic nature, PAs occur in cells in the free form, as well as
soluble/insoluble conjugates (Gemperlová et al. 2006). PAs
can reduce the damage of membrane phospholipids
resulting from increased activity of lipoxygenase under
stress (Lester 2000). PAs are responsible for scavenging
reactive oxygen species (ROS) and can indirectly influence
the activity of catalase, peroxidases or superoxide dismutase. PA catabolism in plants is strongly associated with the
production of hydrogen peroxide, because H2O2 is the
frequent product of the polyamine metabolic pathway
(Moschou et al. 2012). Additionally, PA catabolismderived H2O2 production was proven to induce plant cell
death, which simultaneously is a defense response to abiotic stress (Tisi et al. 2011).
Detoxification of ROS is another important PA function.
A study on tobacco and tomato plants overexpressing a
synthesis enzyme, arginine decarboxylase, showed that
transgenic plants have a higher drought stress tolerance and
drastically reduced ROS levels (Wang et al. 2011). It was
also confirmed that low levels of PAs in soy plants, especially of Put and Spd, are linked to an increased damage
from stress and decreased water content (Nayyar and
Chander 2004). In addition, PAs exert a positive influence
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Acta Physiol Plant (2017) 39:202
on the activity of enzymes of the Halliwell–Asada pathway
that controls non-enzymatic antioxidant content such as
ascorbate and glutathione (Kubiś 2001).
In numerous studies, PA inhibitors were used to modify
the cellular PA content to evaluate their role in various
plant processes (Kaur-Sawhney et al. 2003; Takahashi and
Tong 2015). However, little known about the scope of
inhibitor activity, their transport and metabolism (Sobieszczuk-Nowicka and Legocka 2007). However, the
results of in vivo experiments using this chemical should
be estimated carefully because of their unexpected side
effects (Smith 1990). Treatment with polyamine synthesis
inhibitors reduces the plant’s tolerance to stress, but the
simultaneous application of exogenous PAs and inhibitors
can retain their tolerance (Lee 1997; He et al. 2002). Flores
(1991) showed that the use of arginine and ornithine
decarboxylase inhibitors before osmotic stress stimulates
the accumulation of PAs. Furthermore, the use of an
arginine inhibitor reduces the amount of putrescine in
stressed plants (Aziz et al. 1998). Smith et al. (1985)
showed that long-term treatment with DFMA restrains pea
plant growth. However, Yamamoto et al. (2016) did not
observe significant differences in rice plants growth with or
without PA inhibitor.
The aim of the study was to examine changes in the
content of polyamines under stress conditions and DL-adifluoromethylarginine (DFMA) treatment in yellow lupin
plants subjected to soil drought stress, differing in drought
tolerance, and to determine how these compounds affected
the yield components.
Materials and methods
Plant material
Two cultivars of yellow lupin (Lupinus luteus L.): ‘Morocco 4’, the more tolerant to soil drought, and the more
sensitive cv. ‘Taper’ were tested. Estimation of tolerance to
soil drought stress of the tested cultivars was performed
during the previous experiments (Juzoń et al. 2013).
The seed material was received from the Poznańska
Plant Breeding Ltd., Tulce, Poland. Yellow lupin seeds
were sown into a 3.6 kg mixture of horticultural soil
(Ziemia uniwersalna, Ekoziem, Jurków, Poland) with sand
(2:1 v/v) in pots (50 cm 9 20 cm 9 20 cm). The plants
were grown under conditions attributable to the spring–
summer period (April–September), in an open-air vegetation tunnel protected from rain by gardening foil (Agrimpex Ltd., Jarosław, Poland).
When plants developed five to six leaves, watering of
half of them (30 plants) was restricted for 2 weeks and the
seedlings were subjected to soil drought at 25% field water
Acta Physiol Plant (2017) 39:202
capacity (FWC) for a period of 2 weeks. The rest of the
plants (30 plants) were watered to 70% FWC (control).
Plants after the drought period were watered to 70% FWC
to allow further analysis of the yield components as well as
evaluate the impact of re-watering on the physiological
state of tested plants (data not shown). Water content in the
soil was controlled gravimetrically, including the weight of
plants growing in the pots, and by using a moisture meter
MO750 (Extech Instruments Corporation, USA).
On the first day of drought, 15 plants subjected to soil
drought and 15 well-watered plants (control) were fully
sprayed with an aqueous solution of polyamine biosynthesis inhibitor, DL-a-difluoromethylarginine (DFMA)
(2.5 mL/plant), at a concentration of 0.1 mM.
Relative water content in leaves
Relative water content (RWC) was determined in leaves on
the 1st and 14th day of drought according to Eric et al.
(2005)
using
the
following
formula:
RWC
(%) = (Wf - Wd)/(Wt - Wd) 9 100, where Wf, Wd and
Wt represent fresh weight, dry weight and turgid weight,
respectively. Samples were collected from second, fully
developed leaf from each independent plant in 15
replications.
Polyamine (PA) content
Polyamine analyses in the leaves and seeds of yellow lupin
were made according to Marcińska et al. (2013). The
measurements were performed on the plants’ leaves on the
1st and 14th day of drought. Three to four fully formed
leaves (counting from the top) were used for the analyses.
In seeds, PAs were analyzed after plants’ maturation. The
samples were lyophilized and then homogenized in a ball
mill (MM 400, Retsch, Kroll, Germany). 0.02 g of the
lyophilized leaves and seeds was extracted in 1 mL of 5%
chloric acid (VII) and sonicated for 10 min. Then the
samples were centrifuged at 37,0009g for 10 min and the
supernatant was collected. The extraction was repeated and
the supernatants were combined and mixed. The portion
(200 lL) of combined supernatants were neutralized with
10 lL saturated NaOH. After that 400 lL dansyl chloride
solution (5 mg/mL in acetone) and 200 lL saturated
sodium carbonate solution were added. The samples were
incubated at room temperature overnight. Subsequently,
proline solution (100 mg/mL in water) was added and the
mixture was incubated for 30 min. Finally, dansylated PAs
were extracted in 750 lL toluene. The extraction was made
twice and the upper toluene layers were collected, combined and evaporated under nitrogen (TurboVapÒ LV,
Caliper Life Sciences, USA). The dry residue was dissolved in methanol, filtered through 0.22 lm membrane
Page 3 of 10
202
and analyzed by high-performance liquid chromatography
(HPLC). The HPLC system (Agilent Technologies 1100
series, USA) was equipped with a fluorescence detector
and autosampler, Zorbax Eclipse XDB-C18 4.6 9 75 mm
3.5 lm column (Agilent Technologies, Santa Clara, CA,
USA). The mobile phase was methanol and water under a
linear gradient of 60–95% methanol from 1 to 10 min.
Fluorescence detection was conducted at 365 nm excitation wavelength and 510 nm emission wavelength. The
content of PAs was determined in milligram per 1 gram
DW.
Yield component analyses
At the stage of plant maturity, the number of seeds per
plant, number of seeds per pod, dry weight of seeds per
plant, weight of 1000 seeds and the dry weight of shoot
were determined.
Statistical analysis
A completely randomized design was used to perform the
experiment. There were 12 pots with 60 plants in total. A
replication for each treatment (well-watered plants with
and without DFMA or plants subjected to drought with and
without DFMA) consisted of three pots with five plants.
The results presented in figures constitute the mean values ± standard error based on 15 plant as replicates. Data
were analyzed using two-way ANOVA using a statistical
software package STATISTICA 10.0 (Stat-Soft, USA).
Duncan’s multiple range test at the 0.05 probability level
was used to determine the significance of differences
between cultivars, marked as different letters. Student’s
t test at the 0.05 significance level was also used to compare the means for each treatment.
Results
Relative water content in lupin leaves under soil
drought
Relative water content significantly differed between
treatments, within each genotype, which was confirmed by
Duncan’s multiple range test (Fig. 1). On the 1st day of
drought, cv. ‘Morocco 4’ showed a decrease of 4% of
relative water content (RWC) compared to control plants.
On the 14th day of drought, both cultivars showed a
reduction of RWC, the largest after inhibitor treatment in
‘Morocco 4’ leaves (5%). However in cv. ‘Taper’ comparable decrease of RWC values was determined regardless of the absence or presence of DFMA (4 and 3%,
respectively).
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Acta Physiol Plant (2017) 39:202
Taper
Morocco 4
100
100
a
C
b
RWC [%]
90
b
a
D
a
b
bc
90
b
b
cd
c
c
80
80
70
70
No inhibitor
1 day
No inhibitor
Inhibitor
14 day
No inhibitor
1 day
No inhibitor
Inhibitor
14 day
Fig. 1 Effect of soil drought on the relative water content (RWC)
(%) in the leaves of two cultivars of yellow lupin (‘Morocco 4’,
‘Taper’) on day 1 and 14 of drought; C control, 70% FWC, D drought,
25% FWC; the mean values, n = 15. Significant differences according to Duncan’s test at the 0.05 level of probability are marked with
different letters
White-colored bars represent well-watered (control)
plants without and with application of DFMA. Inhibitor of
polyamine biosynthesis was applied exactly on the 1st day
of drought. Since it is difficult to expect measurable effects
of its actions on the day of application, its impact on the
tested parameters was considered only after 14 days of
drought.
Spd increased by 17%. Spm content did not show statistically significant changes both on day 1 and 14, and its
content in the leaves ranged from 20.5 to 34.8 lg g-1 DW.
Agm was not detected in the leaves of the tested species.
The composition of PAs in the leaves was not the same
as in the seeds of yellow lupin. The presence of four PAs:
Agm, Put, Spd and Spm was confirmed in the seeds
(Table 1). Most of all, the seeds contained 1.5-fold less
PAs than the leaves under control conditions. Yellow lupin
seeds obtained from plants growing under control conditions contained the highest amounts of Spm (92.3 lg g-1
DW), nearly fourfold more than in the leaves. The amount
of Agm and Spd was comparable (50.7 and 43.3 lg g-1
DW, respectively), while Put was present in the smallest
quantity (5.83 lg g-1 DW). Moreover, soil drought stress
decreased the PA contents in the seeds, as opposed to an
increase observed in the leaves. The total PA contents in
the seeds declined by 54%. The highest reduction was
observed in Agm and Spm (64 and 65%, respectively),
while Spd showed a 27% decrease. The only exception was
Put that did not show significant changes.
Polyamine (PAs) content in lupin leaves and seeds
under soil drought
Qualitative and quantitative analyses of PA contents were
conducted on the basis of retention times (Rt) of PA
standards (Sigma-Aldrich, Germany) and their peak areas
(Fig. 2). In the analyzed plant material, seven PAs were
identified: agmatine (Agm; Rt = 7.625 min), 1.3-diaminopentane (1.3-dPen; Rt = 8.852 min), putrescine
(Put; Rt = 9.123 min), cadaverine (Kad; Rt = 9.536 min),
1.6-diaminohexane (1.6-dHex; Rt = 10.076 min), spermidine (Spd; Rt = 12.117 min) and spermine (Spm;
Rt = 13.887 min). Among the measured PAs, Put, Spd and
Spm were present at the highest concentrations. An
increase in the total PA content was found in yellow lupin
leaves under drought conditions (Table 1). PA concentrations on the 1st day of drought was 433.7 lg g-1 DW;
thus, there was an increase of 7% compared to control
conditions. Although yellow lupin leaves contained less
PAs on day 14 of drought than on day 1, an increase of
19% compared to control plants was also observed. On the
1st day of drought, lupin leaves showed the highest amount
of Put (170.9 lg g-1 DW) and Spd (184.8 lg g-1 DW),
which accounted for 82% of the total PA content. On that
day, an increase in Put and Spd contents (67 and 11%,
respectively) was also reported. However, on the 14th day
of drought, Put content did not change significantly, while
123
The influence of DFMA on the PA content and yield
components of lupin
The results of DFMA polyamine biosynthesis inhibitor
application are presented in Fig. 3. The two tested cultivars
of yellow lupin demonstrated different reactions to soil
drought and the presence of DFMA. On the 1st day, the
total PA contents did not change significantly in the tolerant cv. ‘Morocco 4’, while an increase by 35% was
detected in the sensitive cv. ‘Taper’. Furthermore, cv.
‘Morocco 4’ had a fivefold higher concentration of 1.3dPen than cv. ‘Taper’ under drought conditions. On the
other hand, cv. ‘Taper’ contained Put under drought, while
Acta Physiol Plant (2017) 39:202
202
Page 5 of 10
Fig. 2 Chromatograms of polyamine pure standard mixture (a) and endogenous polyamines in yellow lupin leaves cv. Morocco 4 at 14 days of
drought without DFMA (b)
Table 1 Effect of soil drought on the total content of polyamines
(lg g-1 DW) and the contents of Agm, Put, Spd and Spm, the most
abundant in the leaves of yellow lupin on the 1st (C1, D1) and 14th
Polyamines (lg g-1 DW)
(C14, D14) day of drought and in mature seeds (C, D); C—control
70% FWC, D—drought, 25% FWC; the mean values, n = 9, nd—not
detected
Leaves
Seeds
C1
D1
C14
D14
C
D
403.2
433.7*
294.4
365.0*
195.9
89.7*
Agmatine
nd
nd
nd
nd
50.7
18.2*
Putrescine
55.9
170.9*
79.8
77.0ns
5.8
4.6ns
Spermidine
207.2
184.8*
163.0
195.7*
43.3
31.7*
Spermine
34.8
32.9ns
24.1
20.5ns
92.3
32.3*
Total content
The most abundant
ns no significant differences
* Statistically significant differences according to Student’s t test at the 0.05 significance level
this polyamine was not found in cv. ‘Morocco 4’. Both
cultivars contained similar concentrations of 1.6-dHex,
ranging from 89.45 to 138.50 lg g-1 DW, but an increase
by 27% was observed in cv. ‘Taper’. Although this cultivar
contained 28% less of Spd than cv. ‘Morocco 4’, it showed
a 43% increase compared to control conditions. Spm
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202 Page 6 of 10
Taper
800
1000
C
ab
600
cd
c
c
cd
200
0
[µg g DW]
-1
c
c
d
[µg g DW]
0
50
50
40
40
[µg g DW]
nd
nd
b
b
cd
c
c
a
a
a
nd
a
30
b
c
20
150
10
nd nd
nd
0
200
ab
a
150
100
c
a
b
cd
d
d
50
c
0
300
ab
bc
a
bc
250
cd
d
200
150
100
100
50
50
0
0
b
ab
200
150
c
d
50
50
a
40
cd
20
100
250
[µg g DW]
b
a
30
0
300
-1
50
0
50
[µg g DW]
nd
100
100
-1
-1
ab bc
150
150
0
200
1.6-diaminohexan
bc
b
nd nd
-1
c
200
200
10
Spermidine
a
cd
0
250
a
50
Spermine
400
200
nd
a
ab
30
40
c
cd
e
20
a
a
30
ab
c
d
e
20
10
10
0
0
No inhibitor
1 day
123
800
600
400
250
Putrescine
D
b
-1
[µg g DW]
Total content of polyamines
Morocco 4
1000
1.3-diaminopentan
Fig. 3 Effect of soil drought on
the total content of polyamines
(lg g-1 DW) in the leaves of
two cultivars of yellow lupin
(‘Morocco 4’, ‘Taper’) on day 1
and 14 of drought; C control,
70% FWC, D drought, 25%
FWC; the mean values, n = 9;
nd not detected. Significant
differences according to
Duncan’s test at the 0.05 level
of probability are marked with
different letters
Acta Physiol Plant (2017) 39:202
No inhibitor
14 day
Inhibitor
No inhibitor
1 day
No inhibitor
14 day
Inhibitor
Acta Physiol Plant (2017) 39:202
Page 7 of 10
concentration in both cultivars did not exhibit any significant changes.
The period of 14 days of soil drought did not affect the
total content of PAs in yellow lupin leaves. Cv. ‘Morocco
4’ contained 1.3-dPen under drought, while its presence in
cv. ‘Taper’ was not confirmed. There was an increase in
Put content in both cultivars: ‘Morocco 4’—25%,
‘Taper’—52%. Cultivar ‘Morocco 4’ was also characterized by a 42% increase of 1.6-dHex, and a 12% decrease of
Spd. Cultivar ‘Taper’ did not show significant modification
in the contents of these PAs. As regards the Spm content, a
decrease was recorded in both cultivars tested (‘Morocco
4’—20%, ‘Taper’—40%).
The application of DFMA on day 14 of drought did not
induce significant changes in the total PA contents in yellow
lupin plants. However, a twofold increase in 1.3-dPen and a
decrease in Spm content in cv. ‘Morocco 4’ was found (50
and 41%, respectively). Other PAs tested were not influenced by DFMA, while Put was not detected at all. Cultivar
‘Taper’ showed a decrease in the Spd and Spm content (13
and 19%, respectively). DFMA did not affect the synthesis
of Put and 1.6-dHex, and 1.3-dPen was not found.
When the inhibitor was not applied, soil drought did not
cause significant changes in yield components (Table 2).
The use of DFMA under drought conditions led to a
reduction in the number of pods per plant, number of seeds
per plant and dry weight of seeds in cv. ‘Morocco 4’ (64,
50 and 54%, respectively). ‘Taper’ was characterized by a
decrease in the number of pods per plant and number of
seeds per pod (32 and 27%, respectively), which was
nearly twofold smaller. The only exception was the weight
of 1000 seeds which was increased by 21% in cv. ‘Morocco 4’.
Table 2 Effect of soil drought
on yellow lupin yield
components (C—control, 70%
FWC; D—drought, 25% FWC),
the mean values, n = 15
Yield component
Number of pods/plant
Number of seeds/pod
Number of seeds/plant
DW of seeds/plant (g)
Weight of 1000 seeds (g)
DW of shoot (g)
202
Discussion
Approximately, one-third of the world’s cultivated land is
affected by the problem of drought stress (Massacci et al.
2008). Drought as one of the major abiotic stresses limits
crop yields all over the world (Sharp et al. 2004). Despite
many studies on morphological, physiological, biochemical
and molecular mechanisms of adaptation, plant tolerance to
drought stress is not well understood, and obtaining
drought-tolerant cultivars is a fairly slow process (Cabuslay
et al. 2002).
In our study, we applied drought stress at the initial
growth phase of lupin to check how it affects the yield of
the tested cultivars. The cessation of watering was introduced when the plants had five to six leaves. We established two measurements days, 1 and 14, which meant,
respectively, the beginning and the end of drought treatment. Rosales et al. (2012) studying drought stress in
common bean measured RWC for the first time 29 days
after sowing and the final measurement was done on the
last day of drought treatment. In their studies, it was
noticed that leaf RWC generally decreased with time in
both well-watered and drought treatments. However, under
drought, RWC of many genotypes were significantly lower
compared to the control condition, and the differences
became larger with time. Also, Subbarao et al. (2000) in
pigeonpea measured RWC not on the first day of drought,
but 10 days after beginning treatment. In our experiment,
control plants throughout the experiment were watered to
70% FWC. A statistically significant decrease in RWC of
control plants at 14 day compared to 1 day could be an
effect of additional environmental factors, and a period of
13 days between these two measurements seems to be
Cultivar
No inhibitor
Inhibitor
C
D
C
D
Morocco 4
2.2bc
2.5bc
4.5a
1.7d
Taper
3.0bc
2.5bc
2.5bc
1.7d
1.7bc
Morocco 4
2.6ab
3.0a
2.5ab
Taper
2.5ab
2.7ab
3.0a
2.2b
Morocco 4
11.0a
10.3ab
10.3ab
5.2d
Taper
7.3c
7.9c
6.5cd
5.3d
Morocco 4
1.0ab
1.1ab
1.3a
0.6bc
Taper
0.6bc
0.8bc
0.6bc
0.4c
Morocco 4
97.7c
99.9bc
95.8bc
121.9a
Taper
88.8c
96.9bc
84.1c
84.9c
Morocco 4
1.8a
1.6ab
1.6ab
1.3ab
Taper
0.9b
0.9b
0.9b
0.7b
Significant differences according to Duncan’s test at the 0.05 level of probability are marked with different
letters
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202 Page 8 of 10
sufficient to observe permanent changes in RWC (Fig. 1).
According to the scheme of the experiment, 14 days of
drought occurred in May when higher temperature
(reaching ca. 15 °C), compared to 1 day, could lead to the
observed decrease. Low air saturation and accompanying
wind (plants grown in an open-air vegetation tunnel) could
be probably responsible for these changes. The important
point is that the differences between well-watered and
plants subjected to drought at 14 day were observed.
PAs are marked with great interest, due to a wide
spectrum of their biological effects. Their action can vary
substantially, depending on the plant species (Zapata et al.
2004). In many cases, stress is associated with an increase
in the free PA content, demonstrating that its metabolism is
a major component of stress tolerance mechanisms. Generally, it is observed that tolerant genotypes accumulate
greater amounts of PAs than the sensitive ones (Hatmi
et al. 2015). The content of free PAs depends on their
biosynthesis as well as on their transport, conjugation and
degradation. In our study, the total PA contents also varied
in cultivars differing in tolerance to soil drought stress. The
more tolerant cv. ‘Morocco 4’ had a higher amount of PAs
compared with the less tolerant cv. ‘Taper’. Put, Spd and
Spm are among the most prevalent PAs in plants (Marcińska et al. 2013), which was also confirmed in the present
study where these three compounds constituted over 90%
of the total PA contents in yellow lupin leaves and seeds.
According to Hura et al. (2015), PA contents may differ
between and within species and rely on plant organ and
developmental stage. In our study, the absence of Put on
the 1st and 14th day of drought could be explained by the
fact that this polyamine is used for further synthesis of Spd
and Spm, of which large amounts were recorded on these
days. Thus, Put was mostly used in the Spd and Spm
synthesis and the remaining amount was not sufficient to be
detected. It was observed that yellow lupin plants were
characterized by the highest amount of Spd in leaves when
compared with Put and Spm; thus, it is presumed that this
triamine can be involved in the tolerance of yellow lupin to
soil drought. Furthermore, Spd seems to be more
stable under drought conditions and in the presence of
DFMA. Therefore, its greater involvement in the plant
response to applied stress is supposed. Moreover, the tolerant cv. ‘Morocco 4’ showed nearly a twofold higher
content of Spd in leaves compared to the sensitive cv.
‘Taper’. However, González de Mejı́a et al. (2003) showed
that the concentration of Spm was higher than that of the
other PAs in bean genotypes, which suggested that Spm, as
opposed to Put, was not used in the biosynthesis of other
PAs.
In our investigations, changes noticed in the content of
PAs in the leaves and seeds of yellow lupin plants showed
opposite trends. While there was an increase in the PA
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Acta Physiol Plant (2017) 39:202
content in the leaves, a decrease was observed in the seeds.
Furthermore, we detected the presence of Agm in yellow
lupin seeds, which was not found in leaves. Spm was a
polyamine that occurred in yellow lupin seeds in the largest
quantities and was drastically decreased under soil drought
conditions. González de Mejı́a et al. (2003) in tepary bean
(Phaseolus acutifolius) seeds obtained similar results,
where Spm concentrations were the highest compared to
that of the others polyamines. Their results imply that Spm
is not used, as Put is, in the biosynthesis of other polyamines. Endogenous PA levels in plants show dynamic
changes under abiotic stress treatments. In the tested leaves
of yellow lupin, Put was elevated on day 1 and did not
show substantial changes on day 14, while Spd was
decreased on day 1 and increased on day 14 of drought.
Similarly, in Arabidopsis, Put increased after drought stress
treatment for 12 days, while Spd declined after 7, 10 and
12 days of drought stress treatment (Alcázar et al. 2010).
To date, several chemicals have been recognized as inhibitors of polyamine biosynthesis (Kaur-Sawhney et al.
2003), including DL-a-difluoromethylarginine (DFMA) and
difluoromethylornithine (DFMO). The experiments conducted using exogenous PA application and/or inhibitors of
enzymes involved in PA biosynthesis indicated a possible
role of these compounds in plant adaptation to several
environmental stresses (Alcázar et al. 2006). Despite the
fact that treatments with PA biosynthesis inhibitors
diminished stress tolerance, this effect was reversed by
simultaneous application of exogenous PAs. However,
these inhibitors are heterogeneous and possess unspecific
roles in PA biosynthesis (Shi and Chan 2014). The current
study showed that the DFMA treatment on the 14th day of
drought decreased the total PAs and Spm content in
‘Morocco 4’ cultivar as well as Spd and Spm in ‘Taper’
cultivar, which made it difficult to clearly identify the
mode of DFMA action in yellow lupin plants. Alcázar et al.
(2006) reported that the stability and specificity of inhibitors were questionable.
Most studies focused on PA metabolism under stress
conditions, excluding their impact on yield. However,
according to Alcázar et al. (2006), manipulation of polyamine biosynthesis by using inhibitors of their biosynthesis
may improve plant tolerance against multiple environmental stresses. In accordance with our research, it seems
that these compounds may exert an indirect impact on yield
components of yellow lupin plants (Table 2). Tolerant
cultivar ‘Morocco 4’ under soil drought condition and
influence of PA inhibitor showed a decline in the Spm
content accompanied also by a decrease of some yield
components. Under soil drought and the influence of
DFMA, ‘Morocco 4’ produced less pods and seeds per
plant and dry weight of these seeds was also reduced.
Probably, this cultivar adapted to more severe hydration
Acta Physiol Plant (2017) 39:202
conditions according to its origin, and in more favorable
conditions can function better even in the presence of
DFMA. Moreover, it is presumed that the concentration of
applied inhibitor was not high enough for Morocco to bring
the expected effect. Sensitive cultivar ‘Taper’, under the
same conditions (drought and DFMA treatments), was
characterized by a decrease in pod number per plant and
seed number per pod. When no inhibitor was used, both
cultivars did not show a significant reduction in the values
of yield components. These results are contrary to our
earlier research, where most of the tested yield components
decreased under drought conditions (Juzoń et al. 2013). It
is assumed that this unexpected situation may be due to the
weather condition, because the year of the conducted
experiment was characterized by high humidity and plants
could not be affected by stress efficiently enough.
Until now, the physiological role of PAs in yellow lupin
tolerance to drought stress is still unclear. Therefore, it
seems necessary to conduct further research that may help
in understanding the mechanisms that could be used in
breeding and farming practice in the future.
Author contribution statement KJ and ES designed and
carried out the experiment, analyzed the data and wrote the
manuscript; IC-M and IM carried out RWC analysis; MD
and PW carried out HPLC analysis. All these authors have
read and approved the final manuscript.
Acknowledgements This work was funded by the National Science
Centre, Poland, Project no. 621/N-COST/09/2010/0.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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