Abstract
Seeds of four lentil genotypes (Castelluccio, Eston, Pantelleria, and Ustica) were subjected to five levels (0, 10, 15, 18, and 21%) of polyethylene glycol (PEG-6000). Germination percentage, root length, tissue water content (WC), α- and β-amylases, α-glucosidase activities, and osmolyte content were evaluated at 24, 48, and 72 h after starting the germination test. Water stress reduced seed germination percentage, root length, and seedling WC in all cultivars to different extent. The increase in proline content and total soluble sugars was greater for Eston and Castelluccio compared to the other genotypes. The activity of the enzymes involved in the germination process decreased in all cultivars; the activities of α-amylase and α-glucosidase were most negatively affected by osmotic stress, mainly in the drought sensitive Ustica and Pantelleria. Overall, Eston and Castelluccio were able to express greater drought tolerance and consequently could be used as a valuable resource for breeding programs.
1. Introduction
Worldwide agricultural productivity is subject to increasing environmental constraints in the form of abiotic stresses that adversely influence plants growth and development causing crop failure and decreasing average yields more than 50% (Buchanan et al. Citation2000; Bartels & Sunkar Citation2005; Mittler Citation2006; Wu et al. Citation2011). In semiarid environments where lentil is widespread, unfavorable soil moisture at sowing often conditions severely seed germination resulting in an irregular seedling emergence, which in turn affects the establishment of a stand, with negative effects on the yield (Mwale et al. Citation2003; Okcu et al. Citation2005). For these reasons, drought tolerance at the germination stage has specific importance moreover in warm environments most vulnerable to climate change (IPCC Citation2007).
Lentil (Lens culinaris L.), one of the oldest domesticated plants in the world, originated from the near East and central Asia, is traditionally cultivated in the Mediterranean basin (Zohary Citation1972). Seeds of this species are an important source of protein for the human diet and the entire biomass of plant is a valued animal feed. Irrigation generally increases lentil yield (Salehi et al. Citation2008), improving seed size, seed yield, biomass yield, and harvest index (Singh & Saxena Citation1990; Silim et al. Citation1993; Khourgami et al. Citation2012). Thus, the successful crop establishment in semiarid areas depends on the rapid and uniform seed germination, which is strictly associated to the ability of seeds to germinate under low water availability (Arjenaki et al. Citation2011). The sequence of events leading to seed germination and root emergence is governed by water uptake from the external medium (Kaur et al. Citation1998; Hodge et al. Citation2009). Water availability plays a significant role in enzymatic reactions, solubilisation and transportation of metabolites, and also as a reagent in the hydrolytic breakdown of proteins, lipids, and carbohydrates in the storage tissues of germinating seeds (Bewley & Black Citation1994; Białecka & Kępczyński Citation2010). Amylase enzymes play an important role during seed germination, hydrolyzing the endosperm starch into metabolizable sugars, which provide the energy for the growth of roots and shoots (Nauriere et al. Citation1992). The activity of such enzymes is reduced by water stress with negative effects on carbohydrate metabolism (Kaur et al. Citation2000; Zeid & Shedeed Citation2006).
Selection of plants with a better drought tolerance is critical in dry environments (Ashraf et al. Citation1992; Tuberosa & Salvi Citation2006). However, controlled and uniformly repeated simulation of drought in the field cannot be easily achieved (Shaheen & Hood-Nowotny Citation2005). The slow progress in developing drought-resistant cultivars also reflects the lack of a specific method for screening the large numbers of genotypes required in breeding for drought (Zeigler & Puckridge Citation1995). Using natural field conditions is difficult because rainfall can eliminate water deficits. However, in vitro drought-screening methods are facilitating progress in our understanding of drought-resistance traits and in our selection of drought-resistant genotypes. Richards (Citation1978) suggested germination as a useful criterion in screening for water stress tolerance. Khakwani et al. (Citation2011) demonstrated that among the six varieties of wheat tested, those who were tolerant to drought during in vitro germination tests were similarly tolerant in field conditions. In addition, Agili et al. (Citation2012) confirmed this finding with experiments on sweet potato. Thus, study of the influence of the drought using osmotic solutions is one of the methods in the evaluation of resistance during the germination phase. Exposure to polyethylene glycol (PEG-6000) solutions has been effectively used to mimic drought stress with limited metabolic interferences as those associated to the use of low molecular weight osmolytes that can be taken up by the plant (Hohl & Schopfer Citation1991). PEG-based in vitro screening for drought tolerance has been proven to be a suitable method to effectively screen large sets of germplasm with good accuracy (Kulkarni & Deshpande Citation2007).
Understanding the biochemical mechanisms involved in plant drought stress tolerance is still a major challenge in biology and agriculture to identify at early stage suitable traits that would support plant breeders in specific selection programs. The main objective of this study was to evaluate the influence of drought stress on seeds of the four cultivars of lentil, which had previously shown to have diverse level of tolerance to NaCl stress (Sidari et al. Citation2008), in order to select the best suitable parents for hybridization in breeding patterns.
2. Materials and methods
2.1. Plant material, germination conditions, and experimental design
The following lentil cultivars were studied in this experiment. Two salt stress tolerant landraces ‘Pantelleria’ and ‘Ustica’: native and cultivated in the homonymous small islands close to Sicily (Southern Italy), a local population ‘Castelluccio di Norcia’: cultivated in Umbria region (Central Italy), and a Canadian commercial variety ‘Eston.’ Seven-month-old seeds (stored at 20±1°C and ±5% R.U.) of each lentil genotype were used. The seeds were selected for size homogeneity, surface-sterilized for 20 min in 30% (v/v) H2O2, rinsed and soaked in distilled water for 1 h. For each of four genotypes, five replicates of 50-seed were placed on a filter paper in 9-cm Petri dishes containing 3 cm3 of distilled water or 10, 15, 18, and 21% of PEG (MW 6000) concentration corresponding to final osmotic potentials of −0.30, −0.51, −0.58, and −0.80 MPa, respectively. We used 10, 15, 18, and 21% of PEG to have an osmotic potential comparable to that of NaCl at the concentrations of 50, 100, 150, and 200 mM that we tested on seed germination of the same cultivars in a previous work (Sidari et al. Citation2008), in order to evaluate similarity or differences in the metabolic traits of salt and drought resistance. The Petri dishes were sealed with Parafilm to prevent evaporation and kept accordingly to a completely randomized design in a growth chamber at a temperature of 25±1°C in the dark with a relative humidity of 70%. Seeds were considered germinated when the radicle had extended for at least 2 mm. The water content (WC) was measured and expressed as a percentage according to the formula WC (%)=(Fresh Weight – Dry Weight/Fresh Weight)×100. Root length (cm) was also measured and for each of four genotypes, five replicates were used.
2.2. Enzyme activity
The activities of α-amylase, β-amylase, and α-glucosidase were determined in the crude extracts of each cultivar. The seeds of each cultivar and for each PEG treatment (0, 10, 15, 18, and 21%) were homogenized in a chilled mortar with distilled water 1:4 (w/v) and centrifuged at 14,000 g for 30 min. The supernatants were filtered through a single layer of muslin cloth and were used for α-amylase (EC 3.2.1.1) (Steup Citation1988), β-amylase (EC 3.2.1.2) (Steup Citation1988), and α-glucosidase (EC 3.2.1.20) (Bergmeyer et al. Citation1983) activity determination.
For α-amylase, a mixture of 3 ml soluble starch (2% v/v) and 3 ml extract was incubated for 60 min at 30°C. After incubation, an equal volume of alkaline color reagent was added to 1-ml incubation mixture, mixed and heated for five min in a boiling water bath. The absorbance at 546 nm was measured against a blank (1 ml H2O plus 1 ml alkaline reagent). The standard curve was obtained by using different concentrations of maltose in the range of 0–1.5 µmol l−1. The alkaline color reagent was prepared by dissolving 1 g of 3,5-dinitrosalycylic acid in a mixture of 40 ml 1 N NaOH solution and 30 ml H2O. Solid potassium sodium tartrate was added and dissolved. The mixture was brought to a final volume of 100 ml (Steup Citation1988). β-amylase was determined as described above but soluble starch was replaced by amylopectin. For α-glucosidase detection, the assay buffer consisted of 50 mM Na-acetate, pH 5.2, containing 10 mM CaCl2. The substrate was 10 mmol l−1 maltose. The samples were incubated for 60 min. The release of glucose was followed by measuring the changes in NADPH at 340 nm in a coupled enzyme reaction of hexokinase and glucose-6-P dehydrogenase. For each treatment, five replicates were used.
2.3. Osmolyte content
To detect free proline content samples (0.3 g) including 5 ml of 3% sulfosalicylic acid were homogenized and centrifuged at 3000 rpm for 20 min. The supernatant was added to 2 ml of glacial acetic acid with 2 ml acidic ninhydrin. The mixture was heated at 100°C for 25 min. After the liquid was cooled, the mixture was added to 4 ml toluene. The absorbance of the extracts was read at 520 nm (Bates et al. Citation1973). The total soluble sugars were determined with the anthrone method (Yemn & Willis Citation1954). For each treatment, five replicates were used.
2.4. Measurement times and statistical analysis
Root length, water content (WC), enzyme activity, and osmolyte content were measured at 24, 48, and 72 h after the start of the test. The data were statistically analyzed separately for each time by a two-way ANOVA according to the adopted experimental design combining PEG concentrations and genotypes. The germination percentage data were previously subjected to arcsine transformation and were reported in tables as untransformed values. The differences between the means were compared by the least significant difference (LSD) test (p≤0.05).
3. Results
Germination was significantly affected by the osmotic potential, by cultivars and their interaction (). Germination of all cultivars started 24 h after sowing. The final germination percentage of the control (0% PEG) reached 100% for each cultivar but with different time. An increase in PEG stress markedly decreased the germination percentage of all cultivars compared to their relative controls. Seventy-two hours after sowing, the germination percentage of Castelluccio and Eston at the highest PEG concentrations (18% and 21%) was higher than that of Pantelleria and Ustica.
Table 1. Germination (%) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C) after 24, 48, and 72 h.
The effects of drought stress, cultivars, and their interaction were also significant on root length. Root length decreased, increasing water stress and time but with different extent (). By increasing PEG concentrations a different behavior among the cultivars was observed. Eston and Castelluccio showed a greater radicle elongation especially at 72 h in comparison to Pantelleria and Ustica. The greatest radicle reduction was observed in Ustica and Pantelleria at 48 and 72 h in presence of PEG at the concentrations of 18 and 21%.
Table 2. Root length (cm) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C) after 24, 48, and 72 h.
In all cultivars, in the absence of stress, the WC increased over time (). The increase in both duration and intensity of osmotic stress caused a gradual decrease in the WC in each cultivar compared to controls. The presence of PEG at different concentrations differently affected the cultivars over time. Increasing the duration of stress and the time, the WC decreased in Pantelleria and Ustica and increased in Eston and Castelluccio. The lowest WCs were detected at 72 h in Ustica and Pantelleria in presence of PEG at the highest (18% and 21%) concentrations.
Table 3. WC (%) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C) after 24, 48, and 72 h.
Total soluble sugar content decreased during the experimental time in Pantelleria and Ustica seeds. On the contrary, as a consequence of the increasing drought stress and time, in Eston and Castelluccio, a gradual increase in total soluble sugars was observed ()
Significant increase in free proline content was also observed in seeds of all genotypes under water stress. Eston and Castelluccio under drought conditions, accumulated more proline than Pantelleria and Ustica ().The highest amount of proline was detected in the above two cultivars in presence of PEG at the concentrations of 18 and 21% at 72 h.
The values of α-amylase, β-amylase, and α-glucosidase activities were constitutively different in the seeds of the four cultivars (–).The activity of these enzymes decreased in a dose dependent manner, differing among the cultivars. The activities of α-, β-amylase, and α-glucosidase in stressed seeds of Eston and Castelluccio were higher, compared to Ustica and Pantelleria with respect to time and stress level. The activities of these enzymes showed a greater decreasing trend in Pantelleria and Ustica in presence of PEG at the concentrations of 18% and 21%, already 24 h after sowing. Among the enzymes involved in the germination process, α-amylase and α-glucosidase were the most negatively affected by drought stress especially in Ustica and Pantelleria cultivars.
Table 4. α-amylase activity (µmoles of reducing sugars formed min−1 g−1 f.w.) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C) after 24, 48, and 72 h.
Table 5. β-amylase activity (µmoles of reducing sugars formed min−1 g−1 f.w.) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C).
Table 6. α-glucosidase activity (µmoles of reducing sugars formed min−1 g−1 f.w.) of lentil seeds as affected by genotype (G), PEG concentration (C) factors and their interaction (G×C) after 24, 48, and 72 h.
4. Discussion
Drought is a multifaceted stress condition that causes serious crops yield limitations depending on plant growth stage, stress duration, and severity. Germination is the most critical and sensitive stage in the life cycles of plants (Ahmad et al. Citation2009) and the seeds exposed to unfavorable environmental conditions such as drought may compromise the subsequent seedling establishment (Albuquerque & Carvalho Citation2003; Soleymani et al. Citation2012). Genetic variability within a species offers a valuable tool for studying mechanism of drought tolerance. Our results highlighted significant differences among the cultivars exposed to drought stress with a remarkably decreased and delayed germination in Ustica and Pantelleria. These results are consistent with those of other studies that have reported that high concentrations of PEG reduce the final germination percentages of lentil (Siahsar et al. Citation2010; Jamaati-e-Somarin & Zabihi-e-Mahmoodabad Citation2011). In Mediterranean semiarid regions, the topsoil WC during the dry season is drastically reduced, sometimes to less than 1%, which is very close to the permanent wilting point estimated for xerophytes (Larcher Citation1995; Padilla & Pugnaire Citation2007; Munodawafa Citation2012). Consequently, plant traits related to water uptake are of paramount importance for explaining plant persistence in Mediterranean-type ecosystems (Valladares et al. Citation2004). The ability to develop extensive root systems contributes to differences among cultivars for drought tolerance and root length is considered an important trait in selection of drought resistant cultivars (Turner Citation1997; Abd Allah et al. Citation2010). Thus, root morphology and/or growth rate may be instrumental to select drought tolerant varieties (Wahbi & Gregory Citation1995; Malik et al. Citation2002). In our study, total root length decreased with increasing PEG concentrations more in Pantelleria and Ustica than in the other two genotypes, indicating a higher sensitivity to osmotic stress in these two landraces. The root length reduction in Pantelleria and Ustica under drought stress may be associated to a reduced cellular division and elongation during germination (Frazer et al. Citation1990). This hypothesis is supported by the results of enzymatic activities involved in the germination process and by the results of WC. Water availability is usually the limiting factor for the germination of non-dormant seeds, affecting the percentage, speed, and uniformity of emergence (Marcos-Filho Citation2005; Kaydan & Yagmur Citation2008). A threshold level of hydration is required for the synthesis of hydrolytic enzymes which are responsible for the hydrolysis of stored substrates. The hydrolyzed products are utilized in seedling tissue synthesis and radicle elongation (Canas et al. Citation2006). In lentil seeds, the role of providing utilizable substrates is taken over mainly by amylases. Inhibition of seed germination (Haouari et al. Citation2013) is directly related to reserve mobilization, energy production through respiration, enzyme and hormonal activity, and dilution of the protoplasm to increase metabolism for successful embryonic growth (McDonald Citation2007). The PEG treatment decreased WC in the seeds of the studied lentil genotypes, as well documented also for other species under similar experimental conditions (Bajji et al. Citation2000; Guoxiong et al. Citation2002; Wu et al. Citation2011; Haouari et al. Citation2013). Seeds of Castelluccio and Eston had higher WC compared to those of Ustica and Pantelleria indicating that the former genotypes have a more efficient water uptake/control system that can be related to the increase in the free proline content and total soluble carbohydrates, suggesting that drought tolerance ability of these two last landraces appears to be associated to the accumulation of osmolytes which improved their water status. The osmolyte content increase is one of the self-defense reactions during water stress in seeds and plants protecting the enzyme system. Drought and salt stress have been reported to limit the mobilization of starchy endosperm reserves in several species, as a result of inhibition of different enzymatic activities (Ashraf & Foolad Citation2005; Besma & Mounir Citation2010; Białecka & Kępczyński Citation2010). Starch mobilization results from simultaneous activities of α-amylase, β-amylase, and α-glucosidase. In germinating seeds, starch degradation is initiated by α-amylase (Kaur et al. Citation2005), that produces soluble oligosaccharides from starch and these are then hydrolyzed by β-amylase to liberate maltose. Finally, α-glucosidase breaks down maltose into glucose, the main respiratory substrate (Sticklen Citation2008), with release of the energy required for essential metabolic functions (Nauriere et al. Citation1992). Consistently, the activity of α- and β-amylases in germinating seeds is reduced by water stress (Zeid & Shedeed Citation2006). Our results confirmed that the amylase activities in lentil seeds decreased under PEG induced drought stress, and suggested that the variation in stress sensitivity of contrasting lentil genotypes may be linked to their ability to osmoregulate under stress, which cause a strong decrease in WC affecting the hydrolytic enzyme activities, particularly α-amylase and α-glucosidase levels highlighting the greatest decrease in the most drought sensitive Ustica and Pantelleria seeds.
The germination of Ustica and Pantelleria, which are previously identified as NaCl resistant genotypes (Sidari et al. Citation2007, Citation2008), was lower than Eston and Castelluccio at the same iso-osmotic PEG concentrations. These results indicate that mechanisms mediating drought stress tolerance at germination stage are different from those that mediate salt stress tolerance (Munns Citation2002). Since PEG does not enter to seeds (Khajeh-Hosseini et al. Citation2003; Mehra et al. Citation2003), these differences can be specifically associated to mechanisms that control ion homeostasis and toxicity (Bohnert et al. Citation1999). Identifying drought resistant cultivars of lentil at early growth stages is essential to cultivate this crop in arid and/or semiarid environments where the survival of other species would be difficult.
Taken together, these findings suggest that seed germination, WC, and root length can be used as traits for rapid selection of drought tolerant cultivars. Eston and Castelluccio can be considered as valuable drought tolerant germplasm. Our data highlighted an opposite response to salt and drought tolerance because the two genotypes resulting resistant to drought (Eston and Castelluccio) were found salt sensitive (Sidari et al. Citation2008) whereas the two drought-sensitive genotypes (Ustica and Pantelleria) were found NaCl tolerant by the same authors. We can conclude that all these lentil genotypes could be used not only in breeding programs to improve tolerance to both drought and salinity stress with the aim to increase the probability of successful legume establishments in arid or semiarid environments but also to be cultivated in environments where water scarcity is a frequent constraint.
Acknowledgments
This research was supported by ‘‘Mediterranea’’ University of Reggio Calabria-Italy, Programmi di Ricerca Scientifica RDB-2011.
Related Research Data
References
- Abd Allah AA, Badawy Shimaa A, Zayed BA, ElGohary AA. 2010. The role of root system traits in the drought tolerance of rice (Oryza sativa L.). World Acad Sci Eng Technol. 68:1378–1382.
- Agili S, Nyende B, Ngamau K, Masinde P. 2012. Selection, yield evaluation, drought tolerance indices of orange-flesh sweet potato (Ipomoea batatas Lam) Hybrid Clone. J Nutr Food Sci. 2:138.
- Ahmad S, Ahmad R, Ashraf MY, Ashraf M, Waraich EA. 2009. Sunflower (Helianthus annuus L.) response to drought stress at germination and growth stages. Pak J Bot. 41:647–654.
- Albuquerque FMCD, Carvalho NMD. 2003. Effect of type of environmental stress on the emergence of sunflower (Helianthus annuus L.), soyabean (Glycine max (L.) Merril) and maize (Zea mays L.) seeds with different levels of vigor. Seed Sci Technol. 31:65–467.
- Arjenaki FG, Dehaghi MA, Jabbari R. 2011. Effects of priming on seed germination of Marigold (Calendula officinalis). Adv Environ Biol. 5:276–280.
- Ashraf M, Bokhari H, Cristiti SN. 1992. Variation in osmotic adjustment of lentil (Lens culinaris, Medik) in response to drought. Acta Bot Neerl. 41:51–62.
- Ashraf M, Foolad MR. 2005. Pre-sowing seed treatment – a shotgun approach to improve germination growth and crop yield under saline and non-saline conditions. Advan Agron. 88:223–271.
- Bajji M, Lutts S, Kinet JM. 2000. Physiological changes after exposure to and recovery from polyethylene glycol-induced water deficit in roots and leaves of durum wheat (Triticum durum Desf.) cultivars differing in drought resistance. J Plant Physiol. 157:100–108. 10.1016/S0176-1617(00)80142-X
- Bartels D, Sunkar R. 2005. Drought and salt tolerance in plants. Critical Rev Plant Sci. 24:23–58. 10.1080/07352680590910410
- Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water stress studies. Plant Soil. 39:205–207. 10.1007/BF00018060
- Bergmeyer HU, Grabl M, Walter HE. 1983. Enzymes. In: Bergmeyer HU, Bergmeyer J, Grabl M, editors. Methods of enzymatic analysis. Weinheim: Verlag Chemie; p. 126–328.
- Besma BD, Mounir D. 2010. Salt stress induced changes in germination, sugars, starch and enzyme of carbohydrate metabolism in Abelmoschus esculentus L. (Moench.) seeds. Afr J Agric Res. 5:1412–1418.
- Bewley JD, Black M. 1994. Seeds: physiology of development and germination. New York: Plenum Press.
- Białecka B, Kępczyński J. 2010. Germination, α-, β-amylase and total dehydrogenase activities of amaranthus caudatus seeds under water stress in the presence of ethephon or gibberellin A3. Acta Biol Cracov Ser Bot. 52:7–12.
- Bohnert HJ, Su H, Shen B. 1999. Molecular mechanisms of salinity tolerance. In: Shinozaki K, Yamaguchi-Shinozaki K, editors. Molecular responses to cold, drought, heat and salt stress in higher plants. Austin, TX: Landes Company; p. 29–60.
- Buchanan BB, Gruissem W, Jones RL. 2000. Biochemistry and molecular biology of plants. Rockville, MD: American Society of Plant Physiologists.
- Canas RA, Canovas FM, Canton FR. 2006. High levels of asparagines synthetase in hypocotyls of pine seedlings suggest a role of the enzyme in re-allocation of seed scord nitrogen. Planta. 224:83–95.
- Frazer TE, Silk WK, Rost TL. 1990. Effect of low water potential on cortical cell length in growing region of maize roots. Plant Physiol. 93:648–651. 10.1104/pp.93.2.648
- Guoxiong C, Krugman T, Fahima T, Korol AB, Nevo E. 2002. Comparative study on morphological and physiological traits related to drought resistance between xeric and mesic Hordeum spontaneum lines in Israel. Barley Gen Newsl. 32:22–33.
- Haouari CC, Nasraoui AH, Carrayol E, Gouia H. 2013. Variations in α-, β-amylase and α-glycosidase activities in two genotypes of wheat under NaCl salinity stress. Afr J Agricult Res. 8:2038–2043.
- Hodge A, Berta G, Doussan C, Merchan F, Crespi M. 2009. Plant root growth, architecture and function. Plant Soil. 321:153–187. 10.1007/s11104-009-9929-9
- Hohl M, Schopfer P. 1991. Water relations of growing maize coleoptiles. Comparison between mannitol and polyethylene glycol 6000 as external osmotica for adjusting turgor pressure. Plant Physiol. 95:716–722. 10.1104/pp.95.3.716
- IPCC. 2007. Climate change 2007: synthesis report, contribution of working groups I, II and III to the fourth assessment report of the intergovernmental panel on climate change. In: Pachauri RK, Reisinger A, editors. Core writing team. Geneva: IPCC; 104 p.
- Jamaati-e-Somarin S, Zabihi-e-Mahmoodabad R. 2011. Evaluation of drought tolerance indices of lentil varieties. Adv Environ Biol. 5:581–584.
- Kaur S, Gupta AK, Kaur N. 1998. Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth. Plant Growth Regul. 25:29–33. 10.1023/A:1005997819857
- Kaur S, Gupta AK, Kaur N. 2000. Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress. Plant Growth Regul. 30:61–70. 10.1023/A:1006371219048
- Kaur R, Liu X, Goerup O, Zhang A, Yuan X, Balk SP, Schneider MC, Lu ML. 2005. Activation of p 21-activated kinase 6 by MAP kinase 6 and p 38 MAP kinase. J Bio Chem. 280:3323–3330. 10.1074/jbc.M406701200
- Kaydan D, Yagmur M. 2008. Germination, seedling growth and relative water content of shoot in different seed sizes of triticale under osmotic stress of water and NaCl. Afr J Biotechnol. 7:2862–2868.
- Khajeh-Hosseini M, Powell AA, Bingham IJ. 2003. The interaction between salinity stress and seed vigour during germination of soybean seeds. Seed Sci Technol. 31:715–725.
- Khakwani AA, Dennett MD, Munir M. 2011. Drought tolerance screening of wheat varieties by inducing water stress conditions. Songklanakarin J Sci Technol. 33:135–142.
- Khourgami A, Maghooli E, Rafiee M, Bitarafan Z. 2012. Lentil response to supplementary irrigation and plant density under dry farming condition. Int J Sci Adv Technol. 2:51–55.
- Kulkarni M, Deshpande U. 2007. In Vitro screening of tomato genotypes for drought resistance using polyethylene glycol. Afr J Biotechnol. 6:691–696.
- Larcher W. 1995. Physiological plant ecology. Berlin: Springer.
- Malik AI, Colmer TD, Lambers H, Setter TL, Schortemeyer M. 2002. Short-term waterlogging has long-term effects on the growth and physiology of wheat. New Phytol. 153:225–236. 10.1046/j.0028-646X.2001.00318.x
- Marcos-Filho JM. 2005. Fisiologia de sementes de plantas cultivadas [Seed physiology of cultivated 480 plants]. Piracicaba: FEALQ.
- McDonald MB. 2007. Seed moisture and the equilibrium seed moisture content curve. J Seed Technol. 29:7–18.
- Mehra V, Tripathi J, Powell AA. 2003. Aerated hydration treatment improves the response of Brassica juncea and Brassica campestris seeds to stress during germination. Seed Sci Technol. 31:57–70.
- Mittler R. 2006. Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11:15–19. 10.1016/j.tplants.2005.11.002
- Munodawafa A. 2012. The effect of rainfall characteristics and tillage on sheet erosion and maize grain yield in semiarid conditions and granitic sandy soils of Zimbabwe. Appl Environ Soil Sci. 2012:1–8. 10.1155/2012/243815
- Munns R. 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25:239–250. 10.1046/j.0016-8025.2001.00808.x
- Mwale S, Hamusimbi C, Mwansa K. 2003. Germination, emergence and growth of sunflower (Helianthus annuus L.) in response to osmotic seed priming. Seed Sci Technol. 31:199–206.
- Nauriere C, Doyen C, Thevenot C, Dauffant J. 1992. β-amylase in cereals: study of the maize β-amylase system. Plant Physiol. 100:887–893. 10.1104/pp.100.2.887
- Okcu G, Kaya MD, Atak M. 2005. Effects of salt and drought stresses on germination and seedling growth of pea (Pisum sativum L.). Turk J Agric For. 29:237–242.
- Padilla FM, Pugnaire FI. 2007. Rooting depth and soil moisture control Mediterranean woody seedling survival during drought. Funct Ecol. 21:489–495. 10.1111/j.1365-2435.2007.01267.x
- Richards RA. 1978. Variation between and within species of rapeseed (Brassica campestris and B. napus) in response to drought stress. III. Physiological and physicochemical characters. Aust J Agric Res. 29:491–501.
- Salehi M, Haghnazari A, Shekari F, Faramarzi A. 2008. The study of seed yield and seed yield components of lentil (Lens culinaris Medik) under normal and drought stress conditions. Pak J Biol Sci. 11:758–62. 10.3923/pjbs.2008.758.762
- Shaheen R, Hood-Nowotny RC. 2005. Effect of drought and salinity on carbon isotope discrimination in wheat cultivars. Plant Sci. 168:901–909. 10.1016/j.plantsci.2004.11.003
- Siahsar BA, Ganjali S, Allahdoo M. 2010. Evaluation of drought tolerance indices and their relationship with grain yield of lentil lines in drought-stressed and irrigated environments. Aust J Basic Appl Sci. 4:4336–4346.
- Sidari M, Muscolo A, Anastasi U, Preiti G, Santonoceto C. 2007. Response of four genotypes of lentil to salt stress conditions. Seed Sci Technol. 35:497–503.
- Sidari M, Santonoceto C, Anastasi U, Preiti G, Muscolo A. 2008. Variations in four genotypes of lentil under NaCl-salinity stress. Am J Agric Biol Sci. 3:410–416. 10.3844/ajabssp.2008.410.416
- Silim SN, Saxena MC, Erskine W. 1993. Adaptation of lentil to the Mediterranean environment. II. Response to moisture supply. Exp Agric. 29:21–28. 10.1017/S0014479700020378
- Singh KB, Saxena MC. 1990. Studies on drought tolerance: Annual report. Aleppo, Syria: ICARDA.
- Soleymani A, Hesam M, Shahrajabian H. 2012. Study of cold stress on the germination and seedling stage and determination of recovery in rice varieties. Int J Biol. 4:23–30.
- Steup M. 1988. Starch degradation. In: Preiss J, editor. The biochemistry of plants: a comprehensive treatise. San Diego, CA: Academic Press; p. 255–296.
- Sticklen MB. 2008. Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet. 9:433–443. 10.1038/nrg2336
- Tuberosa R, Salvi S. 2006. Genomics-based approaches to improve drought tolerance of crops. Trends Plant Sci. 11:405–412. 10.1016/j.tplants.2006.06.003
- Turner NC. 1997. Further progress in crop water relations. Adv Agron. 58:293–338.
- Valladares F, Vilagrosa A, Peñuelas J, Ogaya R, Camarero JJ, Corchera L, Sisó S, Gil-Pelegrín E. 2004. Estrés hídrico: ecofisiología y escalas de la sequía [Water stress: ecophysiology and scales of drought]. In: Valladeres F. Ecología del Bosque Mediterráneo en un Mundo Cambiante. Madrid: EGRAF, SA; p. 163–190.
- Wahbi A, Gregory PJ. 1995. Growth and development of young roots of barley (Hordeum vulgare L.) genotypes. Ann Bot. 75:533–539. 10.1006/anbo.1995.1055
- Wu C, Wang Q, Xie B, Wang Z, Cui J, Hu T. 2011. Effects of drought and salt stress on seed germination of three leguminous species. Afr J Biotechnol. 10:17954–17961. 10.5897/AJB11.1927
- Yemn EW, Willis AJ. 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem J. 57:508–514.
- Zeid IM, Shedeed ZA. 2006. Response of alfalfa to putrescine treatment under drought stress. Biol Plant. 50:635–640. 10.1007/s10535-006-0099-9
- Zeigler RS, Puckridge DW. 1995. Improving sustainable productivity in rice based rainfed lowland systems of South and Southeast Asia. Feeding four billion people: the challenge for rice research in the 21st century. GeoJournal. 35:307–324. 10.1007/BF00989138
- Zohary D. 1972. The wild progenitor and place of origin of the cultivated lentil Lens culinaris. Econ Bot. 26:326–332. 10.1007/BF02860702