FREE PROLINE CONTENT IN LEAVES OF Salix viminalis alix

E C O L O G I C A L C H E M I S T RY A N D E N G I N E E R I N G
Vol. 15, No. 1–2
A
2008
Anna STOLARSKA
STOLARSKA*,, Jacek WRÓBEL
WRÓBEL*
and Krystyna PRZYBULEWSKA
PRZYBULEWSKA**
**
FREE PROLINE CONTENT IN LEAVES OF Salix
alix viminalis
AS AN INDICATOR OF THEIR RESISTANCE
TO SUBSTRATE SALINITY
ZAWARTOŚĆ WOLNEJ PROLINY W LIŚCIACH Salix
alix viminalis
JAKO WSKAŹNIK ICH ODPORNOŚCI NA ZASOLENIE PODŁOŻA
Summary: In the own research, an attempt was undertaken to evaluate resistance of three basket willow
clones – Bjor, Jorr and Tora – to the stress induced by substrate salinity and to determine the content of free
amino acid, ie of proline, in leaves as a sensitive bioindicator in differentiation of stress agent intensity as
well as to measure selected physiological indices.
Hydroponic experiment was carried out in 2004–2005. In this two-factor experiment, the first factor
was three basket willow clones, Bjor, Jorr and Tora, whereas four levels of substrate salinity: 0.00 (control),
0.034, 0.068 and 0.102 mol · dm−3, represented the second factor. During the vegetation period, the intensity
of gaseous exchange, ie E-transpiration (mmol ⋅ m−2 ⋅ s−1) and A-assimilation of CO2 (μmol ⋅ m−2 ⋅ s−1), as well
as water saturation deficit in leaves (WSD) were measured. Basing on the results of gaseous exchange, water
consumption efficiency in photosynthesis ωF [mmol
[mmol⋅mol
mol−1] was calculated. In the plant material (leaves),
proline content [mg ⋅ g−1 d.m.] was determined 7 days after salination of substrate with the method of Bates
et al. (1973), as well as sodium and potassium contents [mg
[mg⋅kg
kg−1] with AAS method and their quantitative
proportions (K/Na ratio) and fresh and dry matters [g].
As a result of the conducted experiment, significant differences were found in the content of free proline
in the leaves of examined clones and between the levels of solution salinity. The leaves of Tora clone were
characterised by significantly smaller concentration of free proline than those of Bjor and Jorr clones. While
referring to salinity level, proline was accumulated by willow leaves most when substrate salinity was the
largest; there was over two times more proline in Bjor and Jorr clones than under control conditions.
The synthesis of that amino acid also significantly depended on the content of Na+ and K+ ions in leaves,
but differently. In Bjor i Jorr clones, it rose together with the increase in the number of Na+ ions, whereas
together with the increase of K+ ions in Tora clone.
In Bjor clone, significant negative relationships were also found between the content of that amino acid and
the volume of fresh and dry matter in the overground part of basket willow and positive relations between
proline and (WSD).
Keywords:: Salix viminalis
Keywords
viminalis,, salinity, proline, assimilation, transpiration
* Department of Plant Physiology, Agricultural University in Szczecin, ul. J. Słowackiego 17, 71-434
Szczecin, Poland, email: [email protected]
** Department of Microbiology and Biotechnology of Environment
140
Anna Stolarska, Jacek Wróbel and Krystyna Przybulewska
Salinity is one of environmental stresses that affect plants more and more frequently.
Excessive concentration of chloride salts in soil results in the occurrence of physiological
drought and disturbances in water and mineral balance of plants, which in consequence
leads to damage of assimilation apparatus [1, 2].
Under increased salinity level in the soil, cell metabolism changes. Cell structure and
their chemical composition are disturbed [3, 4]. Osmotic stress induced by a surplus of
Na+ and Cl− ions in soil hampers plant water intake and maintenance of full cell turgor.
This in turn leads to synthesis of free amino acids in plant, which elevate the ability of
water maintenance in plant cells and take part in regeneration of damaged cells after
stress disappearance. Such a role is just played by proline, which is a main osmoregulator
and a regular component of plant cells, apart from alanine and glycine [5]. It plays
a special role in formation of protective barriers against penetration of “stressor” and
controls morphogenesis process and plant elongation growth [2].
In the opinion of many authors [3, 5], accumulation of free proline is an indicator of
stress intensity and a factor that determines organism repair capabilities. In general, it is
larger in plants that are characterised by larger resistance to stress [6].
Therefore, it appeared necessary to continue research work on determining sensitivity
of different plant species to substrate salinity and their resistance mechanisms as well
as on searching for answers as to whether plant sensitivity to increased concentration
of Na+ and Cl− ions is solely a variety-specific character or to what extent of substrate
salinity the acclimatisation of plants that are not halophytes is possible. Thus attempts
have been undertaken to select plant varieties that are resistant to salt stress [7].
Large adaptive abilities to different environmental conditions are showed among others by aspens and poplars, but in particular by willows, which distinguish themselves by
large number of ecotypes [2, 8]. As a result of breeding and selection work, many hybrid
forms of Salix viminalis have been obtained, which are characterised both by rapid growth
of biomass and large adaptive ability to different environmental conditions. This makes
possible use them not only in plantation culture but also in environmental protection, among
others in soil conservation and degraded land reclamation. The range of the so-called
ecological tolerance of basket willow is very broad [9].
It was also attempted to show whether proline can be a sensitive bioindicator in
evaluating the resistance of examined basket willow clones to salt stress.
Material and methods
Hydroponic experiment, in complete randomisation design in random block model
with four series and three repetitions, was carried out in 2004–2005.
In this two-factor experiment, the first factor was three basket willow clones, Bjor,
Jorr and Tora, whereas four levels of substrate salinity: 0.00 (control), 0.034, 0.068 and
0.102 mol NaCl · dm−3, represented the second factor.
Basket willow cuttings grew in the Hoagland hydroponic substrate until they developed proper root system and were 40 cm high. Thereafter, 2 cuttings of examined basket
willow were placed into each respective NaCl solutions and aerated hydroponic culture.
The experiment was carried on for 40 days.
Free Proline Content in Leaves of Salix viminalis as an Indicator of their Resistance...
141
During the vegetation period, the intensity of gaseous exchange, ie E-transpiration
[mmol ⋅ m−2 ⋅ s−1] and A-assimilation of CO2 [μmol ⋅ m−2 ⋅ s−1], as well as water saturation
deficit in leaves (WSD) were measured four times. For measuring the gaseous exchange,
a portable infrared gas analyser IRGA LCA-4 (ADC Bioscientific Ltd., Hoddesdon,
UK) was used, operating in an open model with PLC-4 type chamber (manufactured
by the same producer). During the measurement, the chamber with a leaf was placed
under a halogen lamp (Xenophot HLX, OSRAM), with an illuminance of about
1000 μmol
mol · m2 · s−1. Basing on the results of gaseous exchange, water consumption
efficiency in photosynthesis ωF [mmol ⋅ mol−1] was calculated. In the plant material
(leaves), proline content [mg ⋅ g−1 d.m.] was determined 7 days after salination of substrate
with the method of Bates et al. [1], as well as sodium and potassium contents [mg ⋅ kg−1]
with AAS method and their quantitative proportions (K : Na ratio) and fresh and dry
matters [g].
The obtained results for proline content in leaves were analysed statistically by
means of analysis of variance, whereas significance of factors was tested using Tukey’s
test at α = 0.05. Also simple regression was calculated between proline content and the
aforesaid parameters.
Results and discussion
The results referring to proline content in the leaves of three Salix viminalis clones
are presented in Table 1 – for main factors, ie clones and salinity, and in Fig. 1 – for
interactions.
Table 1
Mean proline content in leaves of three basket willow clones exposed to salination with sodium
chloride (NaCl)
Clone
Proline content [mg · g−1 d.m.]
Tora
Jorr
Bjor
Salinity NaCl [mol · dm−3]
0.000
0.034
0.068
0.102
0.035 a
0.118 b
0.136 b
0.068 a
0.059 a
0.099 a
0.160 b
a, b – homogenous groups
Significant differences were found in free proline content in the leaves of examined
clones and between NaCl salinity levels. The leaves of Tora clone were characterised by
significantly lower concentration of free proline [0.0347 mg ⋅ g−1 d.m.] compared with
those of Bjor [0.136 mg ⋅ g−1 d.m.] and Jorr [0.119 mg ⋅ g−1 d.m.] clones. On the other
hand, no statistically significant differences were found between Bjor and Jorr clones
142
Anna Stolarska, Jacek Wróbel and Krystyna Przybulewska
Fig. 1. Effect of different substrate NaCl concentrations on proline content in leaves of three clones of Salix
viminalis
(Table 1). This testifies a different sensitivity of the examined willow clones to stressor
intensity, which is frequently determined by individual properties [2, 8] Studies of some
authors [11, 12] suggest that proline level under the same stress conditions can vary
within different species as well as within varieties of the same species, and even within the same organs. Bandurska [12] found significant differences in proline content in
different leaf parts in four barley cultivars.
While referring to salinity levels, most proline [0.160 mg ⋅ g−1 d.m.] was accumulated
by willow leaves when substrate salinity was the highest, and it was twice more than
under control conditions.
When analysing the interaction between salinity levels and willow clones, its significant effect on the synthesis of that amino acid was found (Fig. 1). Under control
conditions, no statistically significant differences in proline concentration were found
between the clones. However, in subsequent concentrations the content of that amino
acid differed significantly. In the leaves of Bjor clone, proline level increased successively together with salinity concentration, whereas in those of Tora clone the level
of that osmoregulator did not take until the salt concentration was the highest, ie
0.136 mol · dm−3 NaCl. On the other hand, in Jorr clone the lowest salt concentration
resulted in a small decrease in proline synthesis in leaves, whereas subsequent salinity
concentrations increased significantly its accumulation (Fig. 1). Most free proline was
synthesised by the leaves of Bjor and Jorr clones under highest salinity conditions, ie
Free Proline Content in Leaves of Salix viminalis as an Indicator of their Resistance...
143
0.21 mg ⋅ g−1 d.m. on the average. There are, however, not many reports on the examined
indicator in woody plants (trees and shrubs). Hollwarth [10] quotes that the amount of free
proline in the leaves of the common horse-chestnut Aesculus hippocastanum increased
clearly after one-week long treatment with NaCl solution.
Highly significant positive correlation was found between proline content and salinity
level in Bjor and Jorr clones, whereas it was non-significant in Tora clone. The highest
coefficient of correlation was found for Bjor clone (r = 0.98) (Table 2).
Table 2
Equations and coefficients of simple correlation describing relationships between proline content
in leaves of three basket willow clones and selected traits
Character (y)
Character (x)
Salinity
Na
K
K/Na
A
Proline
E
ωF
f.m.
d.m.
WSD
Clone
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Tora
Bjor
Jorr
Na – sodium [mg ⋅ kg−1]
K – potassium [mg ⋅ kg−1]
K : Na – potassium : sodium ratio
A – assimilation rate (CO2) [μmol
[ mol ⋅ m−2 ⋅ s−1]
E – transpiration [mmol ⋅ m−2 ⋅ s−1]
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
Regression equation
= 0.02337 +0.00458x
= 0.01363 +0.4901x
= 0.0918 +0.02774x
= 0.06981 –0.00001x
= 0.09099 +0.00003x
= 0.03890 +0.0006x
= −0.02597 +0.00001x
= 0.05304 +0.00001x
= 0.07148 +0.00001x
= 0.2227 +0.0013x
= 0.19152 –0.002x
= 0.14158 –0.0004x
= 0.05540 –0.0043x
= 0.13437 +0.00133x
= 0.11717 –0.0064x
= −0.0432 +0.15826x
= 0.1493 –0.0287x
= 0.11129 –0.0458x
= 0.06944 –0.0037x
= 0.14762 –0.0036x
= 0.13140 –0.0024x
= 0.11461 –0.2069x
= 0.3429 –0.5202x
= 0.11137 –0.0180x
= 0.07961 –0.4470x
= 0.27554 –1.617x
= 0.11987 –0.1192x
= 0.00628 +0.00439x
= 0.22066 +0.0052x
= 0.10999 –0.0003x
Correlation coefficient r
0.10
0.98*
0.63*
−0.47
0.82*
0.78*
0.65*
0.15
0.05
−0.31
−0.97*
−0.45
0.31
0.20
−0.23
0.51
−0.08
−0.10
−0.46
−0.12
−0.24
−0.41
−0.64*
−0.01
−0.28
−0.78*
−0.07
0.48
0.75*
−0.06
ωF – water consumption efficiency in photosynthesis [mmol
[mmol⋅mol
mol−1]
f.m. – fresh matter [g]
d.m. – dry matter [g]
WSD – water saturation deficit in leaves
144
Anna Stolarska, Jacek Wróbel and Krystyna Przybulewska
Proline synthesis depended significantly also on the content of Na+ and K+ ions in
leaves, but not in similar manner. In Bjor and Jorr clones it grew together with the
increase in the number of Na+ ions, whereas in Tora clone together with that in the
number of K+ ions (Table 2). These findings are consistent with the results of study
carried out by Gorham et al. [13], who stated that tolerance to salt stress in a Triticeae
strain was significantly correlated with preferential absorption of potassium cations
over sodium. In the opinion of Karolewski [14], proline content is significantly affected by the content of macro- and microelements in soil. As a result of harmful action of
Na+ ions superabundance in cells, the stress induced by water deficit increases. From
cellular membranes, Ca2+ ions are expelled, bringing about alterations in their permeability and outflow of potassium ions. The increase of Na+/K+ ratio affects unfavourably
the activity of enzymes and hampers protein synthesis [15]. On the other hand, Bandurska [12] stated that more proline is accumulated by the leaves of plants that grew at
high potassium level. The potassium role is supposed to consist in stimulating the activity of arginase, which catalyses the transformation of arginine into proline. The analysis of coefficients of correlation between proline content and the proportion of K+ ions
to Na+ ions in leaves showed significant negative correlation only in Bjor clone.
A negative correlation was demonstrated with sodium ions (Na+), while a positive one
with potassium ions (K+). In Bjor clone, significant negative correlations were also found
between the content of that amino acid and the volume of fresh and dry matter of
the overground part of basket willow and a positive one with water saturation deficit
(WSD). In the opinion of [12], the content of that amino acid is an indicator of intensity
of stress caused by water deficit.
Conclusion
The examined clones of Salix viminalis demonstrated a different proline content and
physiological response to substrate salinity. Under hydroponic salination with sodium
chloride, the leaves of basket willow of Bjor clone were in general characterised by larger content of free proline than those of Jorr and Tora clones. One can thus state that this
amino acid is a good and sensitive bioindicator for the bushy forms of Salix viminalis
under salt stress conditions, while differences in its accumulation in the leaves of examined
basket willow clones may be an indicator of the intensity of stress induced by different
NaCl concentrations in substrate. Furthermore, a negative correlation between leaf proline
content and overground part biomass yield found only in Bjor clone suggests that an
increase in proline content takes place in Bjor clone during salt stress impact with
simultaneous decrease of its productivity. This is connected most likely with the use of
a part of plant energy for the synthesis of free proline, ie of the amino acid that alleviates
effects of salt stress. Attention should be also paid to the fact that this amino acid can be
incorporated as a source of energy for the growth and for the repair of damages during the
post-stress period. Therefore, the experimental results show that Bjor clone is characterised by the highest resistance against stress induced by substrate salinity, whereas Tora
clone by the least one.
Free Proline Content in Leaves of Salix viminalis as an Indicator of their Resistance...
145
Acknowledgements
The paper was supported by the research project BW/IK/03
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ZAWARTOŚĆ WOLNEJ PROLINY W LIŚCIACH Salix
alix viminalis
JAKO WSKAŹNIKI ICH ODPORNOŚCI NA ZASOLENIE PODŁOŻA
St r es zczenie
W badaniach własnych podjęto próbę oceny odporności trzech klonów wierzby wiciowej: Bjor, Jorr i Tora
na stres wywołany zasoleniem pożywki (0.0; 0.034; 0.068; 0.102 mol · dm−3) oraz oznaczenie zawartości wolnego aminokwasu, tj. proliny w liściach jako czułego bioindykatora w różnicowaniu natężenia czynnika stresowego, a także pomiary wybranych wskaźników fizjologicznych.
Dwuczynnikowe doświadczenie hydroponiczne przeprowadzono w latach 2004–2005. W czasie wegetacji mierzono intensywność wymiany gazowej, tj. E-transpiracji (mmol ⋅ m−2 ⋅ s−1) i A-asymilacji CO2
(μmol
mol ⋅ m−2 ⋅ s−1) oraz deficyt wysycenia liści wodą (WSD). Z wyników wymiany gazowej obliczono
ωF – fektywność wykorzystania wody w fotosyntezie [mmol ⋅ mol−1]. W materiale roślinnym (liście), 7 dni po
zasoleniu pożywki oznaczono zawartość proliny metodą Batesa i wspł. (1973) w [mg ⋅ g−1 s.m.], a także zawartość sodu i potasu [mg ⋅ kg−1] (metodą AAS) i ich ilościowe proporcje (K/Na) oraz świeżą i suchą masę [g].
146
Anna Stolarska, Jacek Wróbel and Krystyna Przybulewska
W wyniku przeprowadzonego eksperymentu stwierdzono statystycznie istotne różnice w zawartości wolnej proliny w liściach badanych klonów oraz między poziomami zasolenia roztworów. Liście klonu TORA
charakteryzowały się mniejszą koncentracją wolnej proliny niż liście klonów BJOR i JORR, najwięcej proliny zgromadziły liście wierzby w warunkach największego zasolenia pożywki, u Bjor i Jorr było ponad dwukrotnie więcej proliny niż w warunkach kontrolnych.
Synteza proliny zależała od zawartości jonów Na+ i K+ w liściach, ale niejednakowo, u Bjor i Jorr rosła
wraz ze wzrostem ilości jonów Na+, a u Tora wraz ze wzrostem jonów K+. U klonu Bjor wykazano również
ujemnie zależność pomiędzy zawartością aminokwasu a ilością świeżej i suchej masy części nadziemnej oraz
dodatnią ze wskaźnikiem WSD.
Słowa kluczowe: Salix viminalis
viminalis,, zasolenie, prolina, asymilacja, transpiracja.