The effect of osmotic stress on physiological parameters
in different maize genotypes
Natalija Kravić ([email protected]), Mirjana Vuletić, Ana Nikolić, Vojka Babić,
Danijela Ristić, Vesna Perić and Violeta Anđelković
Maize Research Institute Zemun Polje, S. Bajića 1, 11185 Belgrade, Serbia
Abstract
Drought is a major factor in reducing plants growth, development and productivity. When exposed
to such condition, plants respond by various mechanisms, ranging from whole-plant characteristics
such as life-cycle timing (maturity) and deep root systems to cellular-level functions such as osmoregulation. It seems likely that the cellular-level compounds of drought-tolerance mechanisms are
important and improvement at this level could have a positive impact on whole plant tolerance. After
testing of 6.000 MRI gene bank accessions under controlled drought in Egypt, as well as in Serbia and
Macedonia, four inbred lines differing in drought tolerance under field conditions, were chosen for
laboratory investigations. Genotypes were exposed to 4% polyethylene glycol (PEG) as osmoticum,
at early seedling stage. As growth reduction and accumulation of osmolytes both typically result from
adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation.
To examine this possibility more closely, in this study we analyzed their response to PEG treatment
in respect to root and shoot length, fresh and dry weight and level of proline accumulation. Results
showed growth reduction in all PEG treated genotypes, beeing more pronounced in shoots than in
roots. Proline content increased in all PEG treated genotypes beeing more pronounced in roots.
Key words: growth, maize, polyethylene glycol - PEG, proline, seedling
Introduction
Drought, being the most important environmental abiotic stress, severely impairs plant growth and
development, limits plant performances and productivity, more than any other environmental factor
(Shao et al., 2009). Impacts of drought include growth, yield, membrane integrity, pigment content,
osmotic adjustment water relations and photosynthetic activity (Praba et al., 2009). The susceptibility of plants to drought stress varies in dependence of stress degree, different accompanying stress
factors, plant species and their developmental stages (Demirevska et al., 2009). Acclimation of plants
to water deficit leads to adaptive changes in plant growth and physio-biochemical processes, such as
changes in plant structure, growth rate, tissue osmotic potential and antioxidant defenses (Duan et
al., 2007). Production and accumulation of free amino acids, especially proline by plant tissue under
water deficit conditions is an adaptive response. Proline has been proposed to act as a compatible solute that adjusts the osmotic potential in the cytoplasm (Caballero et al., 2005) and its content can be
used as a physiological marker in relation to osmotic stress. The root is the first organ to be exposed to
water deficit. Leaf growth is very sensitive to water stress, and may be inhibited by a slight reduction
of water potential in the tissue (Hsiau and Xu, 2000). Therefore, under water deficit conditions, it is
assumed that osmotic adjustment in the root occurs before that in the leaf, to enhance turgor pressure
for continued root growth and absorption of water and nutrients. Thus, osmotic adjustment in the
root is expected to delay the onset of water deficit in the shoots, which reduces the activity of stomatal
conductance and photosynthetic activity.
55
The effect of osmotic stress on physiological parameters in different maize genotypes
In order to analyze the responses and adaptation to water deficit, we used four inbred lines with
different drought tolerance under field conditions, to be the subject of this investigation. The intention was to evaluate chosen maize genotypes at early seedling stage for water stress tolerance under
laboratory conditions, using physiological attributes like proline content and parameters of growth.
Materials and methods
Plant material and growing conditions
The study was carried out on three drought tolerant (A1-A3) maize inbred lines (Zea mays L.)
obtained from MRI gene bank collection, selected by visual scoring of drought related secondary
traits and yield characteristics under field conditions and drought sensitive (B 73). Seeds were germinated for three days on moistened filter paper and then transferred into plastic pots containing ¼
strength Knopp solution with modified nitrogen content. The initial pH of the solution was adjusted
to 5.6. Plants were grown for the following six days in a growth chamber under a 12-h photoperiod
at 22/18oC, with the irradiance of 40 Wm-2 and relative humidity of 70 %. For the terminal 48 h of
the growing period, one half of the plants (treatment) were grown on the aerated nutrient solution
supplemented with 4% polyethylene glycol (PEG, Mr 10000), parallel to control plants grown on the
nutrient solution without PEG.
Plant biomass
Plants were uprooted carefully, washed with distilled water and fresh weights of both roots and
shoots recorded. Then, plant samples were oven-dried at 105°C for 24 h up to constant weight and dry
weights have been recorded (Fletcher et al., 1988).
Free proline determination
Proline was extracted and estimated by the method of Bates et al. (1973) and its concentration was
calculated on fresh weight basis and expressed as µg g-1 fresh weight.
Statistical analysis
All analyses were performed in triplicate measurements and the results presented as means ± standard error (SE). Correlation analyses were performed using Pearson’s correlation coefficient.
Results and discussion
Cell growth as one of the most drought sensitive physiological processes due to the reduction in
turgor pressure, is the result of daughter-cell production by meristematic cell divisions and subsequent massive expansion of the young cells. In our study plant growth was estimated by measuring
root and shoot length and fresh and dry weights, which decreased significantly under PEG-induced
osmotic stress as compared to control plants (Table 1). Decrease in length ranged from 1.3 to 40.1% in
roots and from 1.9 to 46.0% in shoots, respectively. Such decline in root and shoot length in response
to drought might be due to either decrease in cell elongation resulting from the inhibitory effect of
water shortage on growth promoting hormones which, in turn, led to a decrease in each of cell turgor,
cell volume and eventually cell growth (Banon et al., 2006), and/or due to blocking up of xylem and
phloem vessels thus hindering any translocation through (Lavisolo and Schuber, 1998).
56
A common adverse effect of water stress on crop plants is the reduction in fresh and dry biomass
production (Zhao et al., 2006). Kamara et al. (2003) revealed that water deficit imposed at various
developmental stages in maize, reduced total biomass accumulation at silking by 37 %, at grain-filling
period by 34 % and at maturity by 21 %. The results of our investigation have shown significant reduction of fresh and dry biomass both in roots and shoots of water-stressed plantlets. Decrease in fresh
weight ranged from 11.0 % up to 62.0 % in roots and from 22.8 % to 64.2 % in shoots, while in dry
weight was in range from 8.5 % to 45.7 % in roots and from 14.4 % to 49.2 % in shoots, respectively.
Such reduction in fresh and dry biomass production of chosen maize inbred lines could be expected
since the early seedling stage is the most sensitive to water deficit.
Table 1. Effect of 4% PEG treatment on growth parameters in root and shoot – lenght, fresh (FW) and dry (DW)
weight of chosen maize inbred lines, expressed as % of change between control and PEG-treated plants
ROOT
Genotype
Length
Fresh weught
SHOOT
Dry weught
Length
Fresh weight
Dry weight
A1
-40.1
-62.0
-45.7
-46.0
-64.2
-49.2
A2
-11.8
-49.5
-22.6
-21.8
-33.8
-23.5
A3
-1.3
-12.1
-8.5
-1.9
-12.3
-14.9
B73
-9.4
-11.0
-14.0
-13.0
-22.8
-14.4
Under water stress conditions, the accumulation of solutes in the cytosol, such as proline, to
lower osmotic potential thereby maintaining cell turgor, is known as osmotic adjustment. Proline
accumulation is the first response of plants exposed to water-deficit stress in order to reduce cells
injury. Progressive drought stress induced a considerable accumulation of proline in water stressed
maize plants. The proline content increase as the drought stress progressed and reached a peak as
recorded after 10 days stress, and then decreased under severe water stress as observed after 15
days of stress (Anjum et al., 2011). In our study after 48-h exposure to PEG-induced osmotic stress,
increase of proline content ranged from 28.5 % up to 150.1 % in roots and from 9.5 % to 36.3 % in
shoots, respectively (Figure 1). Proline can act as a signaling molecule to modulate mitochondrial
functions, influence cell proliferation or cell death and trigger specific gene expression, which can be
essential for plant recovery from stress (Szabados and Savouré, 2009).
Figure 1. Effect of 4% PEG treatment on proline content in roots and shoots of chosen maize inbred lines
Accumulation of proline under stress in many plant species has been correlated with stress tolerance, and its concentration has been shown to be generally higher in stress-tolerant than in stress-sensitive plants. It influences protein solvation and preserves the quarternary structure of complex proteins, maintains membrane integrity under dehydration stress and contributes to stabilizing sub-cellular structures, scavenging free radicals, and buffering cellular redox potential under stress conditions
57
The effect of osmotic stress on physiological parameters in different maize genotypes
(Ashraf and Foolad, 2007). Our results have shown significant negative correlation between changes in
proline content and fresh weight (-0.932) and highly significant negative correlation between changes
in proline content and root length (-0.998). Correlation analysis between % of changes in proline content and growth parameters in shoot has shown no significance (Table 2).
Table 2. Correlation coefficients between % of changes in proline content and observed growth parameters of
chosen maize inbred lines
ROOT
Proline
SHOOT
Length
FW
DW
Proline
1
Length
-0.998
1
FW
-0.932*
0.901ns
1
DW
-0.848
0.820
0.987
Proline
Length
FW
DW
1
**
ns
ns
*
1
-0.061ns
1
-0.220ns
0.974*
1
-0.019
0.998
0.960*
ns
**
1
** - significant at the 0.01 probability level; * - significant at the 0.05 probability level; ns – insignificant
Conclusion
Our investigation has shown that proline accumulation is closely associated with growth inhibition
induced by PEG treatment in respect to root length and fresh weight. Genotype A3, with best field performances according to visual scoring of drought related secondary traits, was found to be the most
tolerant to PEG-induced osmotic stress, exibiting the lowest reduction of observed growth parameters
and the lowest increase in proline content. Hence, proline accumulattion as physiological character
and growth of osmotic stressed seedlings could be applied as effective indices for evaluating maize
genotypes for drought tolerance.
Acknowledgments
This work was supported by Project TR 31028 from the Ministry of Education and Science of Republic of Serbia.
References
Anjum SA, Wang LC, Farooq M, Khan I, Xue LL (2011). Methyl jasmonate-induced alteration in lipid
peroxidation, antioxidative defense system and yield in soybean under drought. J. Agron. Crop Sci.,
197: 296-301.
Ashraf M, Foolad MR (2007). Roles of glycinebetaine and proline in improving plant abiotic stress
resistance. Environ. Exp. Bot., 59: 206- 216
Banon SJ, Ochoa J, Franco JA, Alarcon JJ, Sanchez-Blanco MJ (2006). Hardening of oleander seedlings
by deficit irrigation and low air humidity. Environ. Exp. Bot. 56: 36-43.
Bates LS, Waldren SP, Teare ID (1973). Rapid determination of free proline for water-stress studies.
Plant Soil 39: 205-207.
Caballero JI, Verduzco CV, Galan J, Jimenez ESD (2005). Proline accumulation as a symptom of
drought stress in maize: A tissue differentiation requirement. J. Exp. Bot., 39: 889–897.
58
Demirevska K, Zasheva D, Dimitrov R, Simova-Stoilova L, Stamenova M, Feller U (2009). Drought
stress effects on Rubisco in wheat: changes in the Rubisco large subunit. Acta Physiol. Plant., 31:
1129- 1138.
Duan B, Yang Y, Lu Y, Korpelainen H, Berninger F, Li C (2007). Interactions between drought stress,
ABA and genotypes in Picea asperata. J. Exp. Bot., 58: 3025-3036
Fletcher RA, Santakumari M, Murr DP (1988). Imposition of water stress in wheat seedlings improves
the efficiency of uniconazole induced thermal resistance. Physiol. Plant., 47: 360-364.
Hsiao TC, Xu LK (2000). Sensitivity of growth of root versus leaves to water stress; biophysical analysis
and relation to water transport. J. Exp. Bot. 51: 1595-1616.
Kamara AY, Menkir A, Badu-apraku B, Ibikunle O (2003). The influence of drought stress on growth,
yield and yield components of selected maize genotypes. J. Agric. Sci., 141: 43-50.
Lavisolo C, Schuber A (1998). Effects of water stress on vessel size xylem hydraulic conductivity in
Vitis vinifera L. J. Exp. Bot. 49: 693-700.
Praba ML, Cairns JE, Babu RC, Lafitte HR (2009). Identification of physiological traits underlying
cultivar differences in drought tolerance in rice and wheat. J. Agron. Crop Sci., 195: 30-46.
Shao HB, Chu LY, Jaleel CA, Manivannan P, Panneerselvam R, Shao MA (2009). Understanding water
deficit stress-induced changes in the basic metabolism of higher plants-biotechnologically and
sustainably improving agriculture and the ecoenvironment in arid regions of the globe. Crit. Rev.
Biotechnol., 29: 131-151.
Szabados L, Savouré A (2009). Proline: a multifunctional amino acid. Trends Plant Sci., 15: 89-97.
Zhao TJ, Sun S, Liu Y, Liu JM, Liu Q, Yan YB, Zhou HM (2006). Regulating the drought-responsive
element (DRE)-mediated signaling pathway by synergic functions of trans-active and transinactive
DRE binding factors in Brassica napus. J. Biol. Chem., 281: 10752-10759.
59