The osmotic potential (OP) of solutions of polyethylene glycol (PEG).
Especially over the last 100 years, our unbridled exploitation of the world’s natural resources has severely damaged its vegetation and has also resulted in worrying accumulations of industrial wastes and greenhouse gases. Together, these have upset natural ecosystem balances and have created many environment and climatic problems, including rising temperatures, increasing desertification, serious soil loss, soil salinization and damaging accumulations of soil nitrogen [39, 31, 37]. In many nations, the recent increased incidences of severe drought and associated desertification are coming into especially sharp focus because of their sudden, long term and devastating consequences for the local human population.
Drought imposes one of the commonest and most significant constraints to agricultural production, seriously affecting crop growth, gene expression, distribution, yield and quality [45, 44, 53]. There are numerous reports on photosynthetic and metabolites characteristics under water stress [22, 52, 25, 5]. Generally, photosynthesis is inhibited by water stress, also affects photosynthetic components and chloroplast stress [54, 52]. Plants have evolved a number of mechanisms to adapt to and survive water stress, Some plant species have evolved mechanisms to cope with the stress, including drought avoidance, dehydration avoidance, or dehydration tolerance. Such adaptive mechanisms are the results of a multitude of morphoanatomical, physiological, biochemical, and molecular changes [1, 2, 6]. But to our knowledge, only a few report about the effects of different level water stress on photosynthetic and metabolites of wheat seedlings.
Wheat is an important crop, with some cultivars tolerant to water stress. The purpose of this study was to investigate the effects of water stress on the growth, chlorophyll fluorescence and accumulations of proline, betaine and carbohydrates of wheat seedlings, by using PEG simulated water stress. It was also desired to elucidate mechanisms of water stress damage and to identify possible adaptive mechanisms to water stress. Understanding how wheat manages water stress is important for the reclamation of drought-prone soils and crop production, and possibly also to discover water-stress resistance genes and hence to develop drought-resistance biotechnology in this crop.
2. Materials and methods
2.1. Design of simulated water stress conditions
Water stress conditions were simulated to polyethylene glycol-6000 (PEG) at one of three concentrations: 0, 5, 15 and 25%. The osmotic potentials of the solutions was measured using a water potential meter (Psypro Wescor Corporation, US) . Table 1 results shows how osmotic potential decreases with increasing PEG-6000 concentration.
2.2. Plant materials and growing conditions
Seeds of wheat (
After three days, 25 boxes containing uniform seedlings were selected and randomly divided into five sets of five replicates. One set was used to determine the seedling growth parameters just prior to treatment, a second set was used as the untreated control (0% PEG-6000, watered with Hoagland’s nutrient solution), and the three remaining sets were stressed with one or other of the PEG-6000 solutions. Each PEG subtreatment was applied to a set of five boxes, daily for 7 days.
2.3. Measurement of growth
After the seventh day of treatment, the fresh weights (FW) were recorded after removing surface water by blotting and the dry weights (DW) determined after drying for 15 min in an oven at 80°C and then in a vacuum dryer at 40°C to constant weight. The relative growth rate (RGR) was defined as (ln DW after treatment – ln DW before treatment) / treatment duration. The water content (WC) percentage was calculated as: 100×(FW–DW)/FW .
2.4. Measurement of chlorophyll fluorescence and pigments
The maximal photochemical efficiency of PSII (PSII=Fv/Fm), the photosynthetic efficiency of PSII (Y(II)=Fm’-F/Fm’), non-photochemical quenching (NPQ=Fm-Fm’/Fm’), non-photochemical quenching coefficient (qN=Fm-Fm’/Fm-Fo’), photochemical quenching (qP=Fm’-F/Fm’-Fo’), the efficiency of excitation energy capture by open PSII reaction centers (Fv’/Fm’) and apparent photosynthetic electron transport rate (ETR) were determined between 09:00 and 11:00 h from fully-expanded leaves using an Imaging-PAM (Walz, Effeltrich, Germany], [12, 48]. The leaves were held in the dark for about 20 min before measurement. The intensities of the actinic and saturating light settings were 185μmol/m2s and 2500μmol/m2s PAR, respectively. The contents of carotenoids (Car) and chlorophyll (Chl)
2.5. Measurement of metabolites and organic acids
Proline was extracted with 3% sulfosalicylic acid for 30 min at 70°C and measured with ninhydrin . Betaine was extracted with 80% methanol for 20 min at 70°C and measured as described by Grieve and Grattan (1983). Total soluble sugars (SS) were extracted for 30 min at 70°C in 70% alcohol, and measured using anthrone.
2.6. Measurement of germination
One hundred wheat seeds were germinated on filter paper in germination boxes. The dry seeds were submerged in 100 mL of each of the PEG-6000 solutions described above (with distilled water as the control). The boxes were maintained at 20°C in the dark for 10 d, five replicates of each PEG treatment were prepared. Percentages of germinated seeds were scored daily, based on the emergence of the radicles. The germinative Energy(Ge), germinative Percentage(Gp), and germination activity Index(Ai) of wheat seeds were modified using Ge = n/Nx100% (n: the number of germination of seeds in 4 days; N: the total number of seeds); Gp = nl/Nlx100% (n1: the number of germination of seeds at 10 days; N1: the total number of seeds).
3. Statistical analysis
Statistical analysis included one-way analysis of variance (ANOVA) in SPSS (Version 13.0, SPSS, Chicago, IL, USA) and Duncan’s method to detect differences in physiological parameters in plants under water stress (
The RGR and WC of shoots and roots all decreased with increasing PEG concentration, with the greatest reductions occurring under the highest water stresses (Fig. 1 A - D,
||Decrease in RGR and WC per 1% increment in PEG-6000 concentration|
4.2. Chlorophyll fluorescence and pigments
The Fv/Fm, Y(II), qP and ETR decreased with increasing PEG concentration, while NPQ and qN contents increased significantly, the effects were much more pronounced under high PEG concentration (Table 3;
The contents of proline increased with increasing PEG concentration, with that in the shoot being significantly higher than that in the root (Fig. 2, A and B;
5. Organic acids
The table 5 shows that the trend in changes in Gp and Ge of wheat seeds under water stress conditions was similar; there was a decreased trend with increased PEG-6000 concentration (
|Treatment||Gp (%)||Ge (%)|
PEG is an osmotic agent, which play an important role in the regulation of mineral elements, hormone, protein metabolism and effects of signal transduction [50, 41]. The main function of PEG is to slow down the moisture rate of import and export seeds, which benefit to reduce membrane system injury in process of seed imbibition and repair impaired membrane system [27, 16]. PEG has been widely used in seed priming and simulated water stress test, the wheat seedlings were treated by three different PEG concentrations.
7. Impact of water stress treatments on growth
In plants in general, an appropriate growth strategy is key to fitness in a competitive situation, so too in wheat seedlings, their growth strategy is critical to survival . The RGR value of a plant reflects its vigour and is considered a good index of its exposure to stresses of all sorts [26, 52]. The RGR response of wheat seedlings exposed to increasing PEG concentrations (Fig. 1 A, P ≤ 0.05), revealed a decrease for roots and shoots (Table 2,
7.1. Impact of water stress treatments on chlorophyll fluorescence and pigments
The chlorophyll fluorescence kinetics react to the “intrinsic” characteristic of photosynthesis and can rapidly and sensitively reflect a plant’s physiological status and its relationship with the environment [Huang et al., 2009]. In this study, PSII values decreased with increasing PEG concentration but these began to decline significantly in 15% PEG concentration. The results indicate that photoinhibition occurs under water stress as a result of damage to the reaction center of photosystem II (Table 3,
Chl and Car are the main photosynthetic pigments of plants, so these are good indicators of the photosynthesis capability of a plant. Under water stress, with the exception of Car which barely changed, the contents of Chl
7.2. Impact of water stress treatments on metabolites
Proline and betaine are also known to play important roles in osmotic adjustment with their accumulation under water stress being observed in many species [46, 38]. Here, the results show that, along with a decrease in osmotic potential, the accumulation of free proline and betaine increased significantly both in the roots and the shoots. This increase would lower the osmotic potential [i.e. make it more strongly negative] in the cells which would help to maintain turgor and thus sustain the normal physiological and biochemical processes in the face of drought (Fig. 2,
Soluble sugars are the main osmotic adjustment substances and so are important indicators of drought tolerance. The results show that the soluble sugars contents of wheat seedlings increases under high PEG concentration. This indicates that they may help to regulate and maintain the activity of physiological processes within the plant in a high water-stress environment by raising the osmotic potential of the cells .
7.3. Impact of water stress treatments on organic acids
The accumulation of organic acids is a physiological response of plants to stress, when plants are suffered by water stress, they can through cells apperceive and transmit drought signal . There nearly no impact on the content of organic acids under blew 15% water stress, it decreased significantly under high stress, but in shoot FA completely opposite change (Fig. 3,
7.4. Impact of water stress treatments on germination
Germination is one of the most critical periods in the life cycle of plants. Under water stress, low water potential is a determining factor inhibiting seed germination [51, 43]. The inhibiting action of water stress on the wheat germination was increased with PEG-6000 concentration increasing (Table 5).
In summary, the growth of wheat seedlings was inhibited by water stress, especially in roots. The function of water regulation occurs outside root, or in apoplast of root, or both outside root and in apoplast of root. Therefore, we propose that the water-potential adjustment of the roots may be a key physiological mechanism for wheat resisting water stress. Proline, betaine and soluble sugar content increase to a greater extent in response to water stress, these data suggest that wheat seedlings may initially sense high drought environments, the harmful effects of water stress on the distribution and accumulation of carbohydrates, it was reflecting the specific detrimental effects of a drought environment. With the extension of PEG-6000 concentrations, wheat seedlings photosynthetic electron transport and photosynthetic primary reaction inhibited, heat disseminate which possess photoprotective effect increased. It implies that there was a closed relationship between the effects of water stress on chlorophyll fluorescence parameters of wheat seedlings. These results provide useful data that will facilitate the development of strategies for the creation of engineered wheat varieties that are more tolerant towards water stress.
This work supported by grants from the Project of the National Natural Science Foundation of China (No. 31170303, 30870238, 30871447, 50709040, 31070398). The basic research special fund operations (1610122012001), the international scientific and technological cooperation projects (No. 2010DFB30550).
Abdalla M. M. El -Khoshiban N. H. 2007 The influence of water stress on growth, relative water content, photosynthetic pigments, some metabolic and hormonal contents of two Triticium aestivum cultivars. 3 2062 2074
Ali Q. Ashraf M. 2011 Induction of Drought Tolerance in Maize (Zea mays L.) due to Exogenous Application of Trehalose: Growth, Photosynthesis, Water Relations and Oxidative Defence Mechanism. 197 258 271
Almansouri M. Kinet J. M. Lutts S. 1999 Compared effects of sudden and progressive impositions of salt stress in three durum wheat (Triticum durum Desf.) cultivars. 154 743 752
Armstrong D. P. Westoby M. 1993 Seedlings from large seeds tolerate defoliation better: a test using phylogenetically independent contrasts. 74 1092 1100
Ashraf M. 2010 Inducing drought tolerance in plants: Recent advances. 28 169 183
Ashraf M. Ahmad M. S. A. Öztürk M. Aksoy A. 2012 Crop Improvement Through Different Means: Challenges and Prospects. 1 15
Bjorkman O. Demmi G. B. 1987 Photon yield of O2 evolution and chlorophyll fluorescence at 77K among vascular plants of diverse origins. 170 489 504
Bush J. K. Van Auken O. W. 1991 Importance of time of germination and soil depth on growth of Prosopis glandulosa (Leguminosae) seedlings in the presence of a C4 grass. 78 1732 1739
Chen Y. P. Chen Y. N. Li W. H. Xu C. X. 2009 Effect of high temperature on photosynthesis in populus euphratica under drought condition. 29 3 474 479
Du Y. Huang Z. L. 2008 Effects of seed mass and emergence time on seedling performance in Castanopsis chinensis. 255 2495 2501
Gale K. R. 2005 Diagnostic DNA markers for quality traits in wheat. 41 181 192
Genty B. Briantais J. M. Baker N. R. 1989 The relationship between the quantum yield of Photosynthetic electron transport and quenching of chlorophyll fluorescence. 990 87 92
Harms K. E. Dalling J. W. 1997 Damage and herbivory tolerance through resprouting as an advantage of large seed size in tropical trees and lianas. 13 617 621
He H. Sun C. H. Du W. Li Y. 2006 Effect and evalution of Entomophthora spp. on controlling Aphis glycines. 28 76 78
Huang H. Y. Dou X. Y. Deng B. Wu G. J. Peng C. L. 2009 Responses of different secondary provenances of Jatropha curcas to heat stress. 45 7 150 155
Jiao S. Y. Li Y. Q. Shayila S. H. Chen X. L. 2009 Seeds Germination and Seedling Growth about 3 Pennisetum Ornamental Grasses under Drought Stress. 29 2 0308 0313
Jing J. H. Ding Z. R. 1981 Determining organic acid content.]- In: Boqinnoke, X.H. (ed.): Analysis Method of Plant Biochemistry. 264 267
Johnson G. N. Youn G. A. J. Scholes J. D. et al. 1993 The dissipation of excess excitation energy in British plant species. 16 673 679
Jurado E. Westoby M. 1992 Seedling growth in relation to seed size among species of Arid Australia. 80 407 416
Kerepesi I. Galiba G. Ba´nyai E. 1998 Osmotic and salt stresses induced differential alteration in water-soluble carbohydrate content in wheat seedlings 46 5347 5354
Kerepesi I. Galiba G. 2000 Osmotic and salt stress-induced alteration in soluble carbohydrate content in wheat seedlings. 40 482 487
Krause G. H. Weis E. 1991 Chlorophyll floresence and photosynthesis. 142 313 349
Leishman M. R. Westoby M. 1994 The role of seed size in seedling establishment in dry soil conditions-experimental evidence from semiarid species. 82 249 258
Li Z. J. Luo Q. H. Wu W. M. Han L. 2009 The effects of drought stress on photosynthetic and chlorophyll fluorescence characteristics of Populus euphratica and P.pruinosa. 24 4 5 9
Liu H. Y. Li J. Y. Zhao Y. Huang K. K. 2007 Influence of drought stress on gas exchange and water use efficiency of Salix psammophila growing in five places. 24 6 815 820
Lutts S. Almansouri M. Kinet J. M. 2004 Salinity and water stress have contrasting effects on the relationship between growth and cell viability during and after stress exposure in durum wheat callus. 167 9 18
Ma H. Y. Liang Z. W. 2005 Research progress on improving germination rate of Leymus chinensis. 27 4 64 68
Martin M. Miceli F. Morgan J. A. Scalet M. Zerbi G. 1993 Synthesis of Osmotically Active Substrates in Winter Wheat Leaves as Related to Drought Resistance of Different Genotypes 171 176 184
Paz H. Martinez-Ramos M. 2003 Seed mass and seedling performance within eight species of Psychotria (Rubiaceae). 84 439 450
Maxwell K. Johnson G. N. 2000 Chlorophyll fluorescence, a practical guide. 51 659 668
Naumann J. C. Young D. R. Anderson J. E. 2008 Leaf chlorophyll fluorescence, reflectance, and physiological response to freshwater and saltwater flooding in the evergreen shrub, Myrica cerifera. Environ. 63 402 409
Pawl B. 1998 Cities and economic development: from the dawn of history to the present.
Pilon-Smits E. A. H. Ebskamp M. J. M. Paul M. J. Jeuken M. J. W. Weisbeek P. J. Smeekens S. C. M. Improved performance of transgenic fructan-accumulating tobacco under drought stress. 107 125 130
Qu Y. Y., P. Mu, X. Q. Li Y. X. Tian, F. Wen, H. L. Zhang Li Z. C. 2008 QTL mapping and correlations between leaf water potential and drought resistance in rice under upland and lowland environments. 34 2 198 206
Ralph P. J. Burchett M. D. 1998 Photosynthetic response of Halophila ovalis to heavy metal stress. 103 91 101
Rau S. Miersch J. Neumann D. Weber E. Krauss G. J. 2007 Biochemical responses of the aquatic moss Fontinalis antipyretica to Cd, Cu, Pb and Zn determined by chlorophyll fluorescence and protein levels. 59 299 306
Reich P. B. Tjoelker M. G. Walters M. B. Vanderklein D. W. Buschena C. 1998 Close association of RGR, leaf and root morphology, seed mass and shade tolerance in seedlings of nine boreal tree species grown in high and low light. Funct. 12 327 338
Rengasamy P. 2002 Transient salinity and subsoil constraints to dryland farming in Australian sodic soils: an overview. 42 351 361
Rhodes D. Hanson A. D. 1993 Quaternary ammonium and tertiary sulphonium compounds in higher plants. Annu. Rev. Plant Physiol. 44 357 383
Richards J. F. 1990 Land transformation 163 178
Ross M. A. Harper J. L. 1972 Occupation of biological space during seedling establishment. 60 70 88
Shao H. B. Liang Z. S. Shao M. G. Wang B. C. 2005 Impacts of P EG-6000 pretreatment for barley ( Hordeum vulgare L. ) seeds o n the effect of their mature embryo in vitro culture and primary investigation on its physiological mechanism. 41 73 77
Shen L. M. David M. Joyee G. F. 1990 Influence of drought on the concent ration and distribution of 2,4- diaminaobutyric acid and other free amino acids in tissues of flat pea (Lathyrus sylvestris L.). 30 497 504
Shi G. Y. Liao W. X. Qin L. F. Lu L. L. 2009 PEG simulated water stress effects on physiological and biochemistry indexes of germination of Toona sinensis seeds. 4 142 145
Shi Z. Shi S. Q. Xiao W. F. Qi L. W. 2008 Influence of dehydration on characteristics of chlorophyll fluorescence of detached leaves in Haloxylon ammodendron and Populus euphratica. 21 4 566 570
Stendle E. Peterson C. A. 1998H ow does water get through roots? 49 775 788
Stewart G. R. Lee J. A. 1974 The role of proline accumulation in haloplytes. 120 279 289
Sun L. Liu S. H. Shi X. D. Xiao M. Tang Z. Y. Zhu H. W. J. Z. 2006 ChenSalt-tolerant physiological and biochemical properties of ten Species of chenopodiaceae halophytes growing in deserts, Xi njiang. 23 209 313
Van K. O. Snel J. F. H. 1990 The use of chlorophyll fluorescence nomenclature in plant stress physiology 25 147 150
Verslues P. E. Ober E. S. Sharp R. E. 1998 Root growth an d oxygen relation at low water potentials impact of oxygen availability in polyethylene glycol solution. 116: 1403 1412
Wang J. G. Chen G. C. Zhang C. L. 2002 The effect s of water stress on soluble protein content, the activity of SOD, POD and CAT of two eco types of reeds ( Phragmi tescommunis ). 22 3 561 565
Xu S. G. Wang J. H. Bao L. J. 2006 Effect of Water Stress on Seed Germination and Seedling Growth of Wheat. 34 5784 5787
Yang X. Q. Zhang S. Q. Liang Z. S. Shan Y. 2004 Effects of water stress on chlorophyll fluorescence parameters of different drought resistance waiter wheat cultivars seedlings. 24 812 816
Zhao Y. J. Weng B. Q. Wang Y. X. Xu G. Z. 2009 Plant physio-ecological responses to drought stress and its research progress. 27 2 45 50
Zhang Y. Q. Mao X. S. Sun H. Y. 2002 Effects of drought stress on chlorophyll fluorescence of winter wheat. 10 13 15
Zhou J. L. Tang X. Q. Wang K. C. 2009 Effect of water stress on content of four organic acids in different cultivated populations of Isatis indigotica. China 34 127 131