Nasserghadimi Farshad, Jahanbakhsh Soodabeh, Ghaffari Mehdi, Ebadi Ali

1 Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil 5619911367, Iran

2 Seed and Plant Improvement Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj 1985713133,Iran

1. Introduction

Drought is the most important factor limiting plant growth and crop production around the world (Abedi and Pakniyat 2010). Water stress occurs when the volume of water loss in a plant is greater than the amount of water absorbed from the soil. Long-term water stress affects all plant processes,leading to a reduction in their production (Jafarzadeh et al.2013). Stone et al. (2001) claimed that the key factor determining the yields of crops in semiarid areas is the amount of moisture available in the soil. Iran, with an average rainfall of 240 mm (one-third of the global average),is considered as a dry or semi-dry area (Jajarmi 2009).The identification of the critical stages of watering and the accurate timing of watering are key factors to restore water,improve irrigation operations, and enhance the tolerance of plants to water shortage in agriculture (Ngouajio et al. 2007).Thus, according to the target of cultivation, an appropriate plan should be used in order to consume water optimally instead of through full irrigation. In order to maximize yield and alleviate the adverse effects of drought, plants need to be watered only at critical stages (Kamkar et al.2011). The reduction of water in plant tissues, on one hand, decreases the growth, photosynthesis, intercellular spaces, growth intensifiers, and accumulation of proline.On the other hand, it closes the pores, affects breathing,destroys proteins and enzymes, and produces toxicants,a variety of reactive oxygen, and hormonal disorders such as an increased level of abscisic acid (Shimshi et al. 1992).Since one of the common influences of drought stress is initial oxidative damage, similar to other environmental tensions (Chen 2000), plants form an antioxidant defence system with enzymatic and non-enzymatic mechanisms,e.g., superoxide dismutase, catalase, with a series of non-enzymatic antioxidant compounds like ascorbic acid,glutathione, alpha-tocopherol, and carotenoids (Prasad 2002; Nasibi 2005).

Sunflower (Helianthus annuus L.) is a major source of vegetable oilis of great importance in the world (Arshi 1992). The water needed for sunflower cultivation during the growth period is estimated to be about 500–600 mm.By increasing the amount of available water, dry matter production increases, although plants can adapt themselves to environmental conditions in case of water shortage (Alyari and Shekari 2000). In 2003, sunflower was planted on 70 000 ha in Iran with over 1 286 kg ha–1production, in comparison to over 1 749 kg ha–1world production in that year (FAO 2013).

In a study, drought stress was found to lead to a decrease in the leaf area index, relative water content of leaves, and seed and oil yield, and an increase in the proline contents in leaves (Ghaffari et al. 2014). Similarly, in another study,drought stress had significant adverse impacts on seed and biological yield, and harvest index (Heydari and Karami 2013).

Since Iran mainly has an arid and semiarid climate,it suffers to a great extent from the shortage of water,which in turn is responsible for the reduction in agricultural production. This research aims to study the resistance of different inbred lines of sunflower to water shortage and their morphological and physiological responses to full and limited irrigation.

2. Materials and methods

The study was conducted in the growing season of 2014–2015 in the research field of the Agricultural Research Station in Khoi, Iran. The research field is located at latitude 38 °37′N and longitude 45°15′E, and at an altitude of 1 103 m above sea level. A factorial experiment in randomized complete block design with three replications was used. The factors consisted of 20 inbred lines(BGK 375, BGK 369, BGK 357, BGK 335, BGK 329,BGK 309, BGK 77, BGK 45, BGK 43, BGK 41, BGK 39,BGK 37, BGK 29, BGK 15, RGK 55, RGK 50, RGK 26,RGK 23, RGK 19, BGK 1) with one level and irrigation at two levels (full and limited). Water withholding was imposed in the flowering stages from R4 (initial flowering) to R6(full flowering). The full and limited irrigation (until the R4 level) of seeds sown into nylon bags was done manually three times a week, followed by no watering from R4 to R6 stages, in limited irrigation. The nylon bags were filled with a mixture of field soil, manure, and blown sand (1:1:1), and then diammonium phosphate (100 g), potassium sulphate(100 g), and urea fertilizer (30 g) were added into them at the eight-leaf stage (V8). The next 30 g urea was distributed when the plantlets had 16 leaves.

The studied characteristics included the proline content,the catalase, peroxidase, and polyphenol oxidase activites,the protein content, the seed yield of each plant, and the relative water content. The following equation was used to calculate the relative water content (Levitt 1980):

RWC (%)=(FW–DW)/(TW–DW)×100

Where, RWC is the relative water content; FW, DW, and TW are the fresh weight, dry weight, and inflammation weight of the leaves, respectively.

To measure the proline content, 0.1 g of leaf sample was crushed into a container with 2 mL of sulfosalicylic acid and then was centrifuged for 10 min at 4 000 r min-1at 4°C. After this, 2 mL of this extract was mixed with 2 mL of ninhydrin and 2 mL of glacial acetic acid. The mixture was then dipped into a water bath for 1 h. Then, 4 mL of toluene was added.The mixture was vortexed and the absorbance of upper solution was measured by a spectrophotometer at 520 nm(Beckman Coulter, USA) (Bates et al. 1973).

The polyphenol oxidase activity was measured by mixing 1.5 mL of Tris buffer (pH=7.6) and 0.4 mL of pyrogallol in a plastic stopper tube, followed by the addition of 0.1 mL of the enzyme extract. After vortexing and placing in a water bath for 5 min in the spectrophotometer at a wavelength of 420 nm, the related number was noted down. To measure the catalase activity, 2.5 mL of Tris buffer (pH=7), in addition to 0.3 mL of hydrogen peroxide,was added to 60 mL of enzyme extract in ice bath, and the data were measured by a spectrophotometer at OD240. On peroxidase enzyme, the Tris buffer (pH=7, 0.1 mol L–1) was mixed with hydrogen peroxide (0.05 mol L–1) and pyrogallol(0.01 mol L–1), and 2.5 mL of this solution was mixed with 50 mL of enzyme extract in ice bath and the data were measured by the spectrophotometer at OD425(Kar and Mishra 1976). To determine the total amount of protein in the solution, the Bradford (1976) method was used. The data were analysed with SAS (ver. 9.12) and averages were compared with each other through a multiple range test at 5% level. Figures were drawn using Excel 2010 software.

3. Results and discussion

3.1. Proline content

According to the results of the analysis of variance, the main effect of irrigation and inbred lines and the interaction effect between irrigation and lines had a significant effect on the proline content at 1% level (Table 1). In this experiment,the proline content in limited irrigation increased by 43.5%in comparison to the increase in full irrigation (Table 2).The mean comparison of the mutual effects showed that the highest proline content was found in BGK 37 under limited irrigation conditions (3.08 mg g–1). The level of this characteristic in this line increased by 49.51% under limited irrigation condition compared to full irrigation. The lowest proline content was seen in BGK 15 in full irrigation condition(1.16 mg g–1) (Table 3).

In general, the survival of a plant under drought stress requires it to be resistant against severe osmotic conditions.Osmoregulation is an important part of the drought tolerance mechanism in plants (Omidi 2010). It seems that proline accumulation is a common response to drought. Under drought stress, the proline accumulation protects plant cells by increasing osmotic pressure (Mohamadet al. 2000;Chaves et al. 2002; Cechin et al. 2010). The increased proline content in sunflower, under drought stress, has already been reported (Unyayar et al. 2004; Ghaffari et al.2014). Osmoregulation, under drought stress in sunflower,prevents a drop in photosynthesis and yield efficiency, by keeping the cells swollen (Rauf 2008).

3.2. The catalase activity

According to the results of the analysis of variance, the main effect of irrigation and inbred lines and also the interaction effect between irrigation and inbred lines had a significant effect on the catalase activity at 1% level (Table 1). The catalase activity in limited irrigation increased by 87.9%in comparison to that in full irrigation (Table 2). The mean comparison of interaction effect showed the highest catalase activity in BGK 39 under limited irrigation condition (49.13 mg g–1), rising by 114.35% compared to full irrigation. However,the lowest level of activity of this enzyme was observed in BGK 309 in full irrigation (Table 3).

The synthesis of a few enzymes during the drought,namely catalase, is considered as an adaptation response to oxidative tension (Mittler 2002), such a rise has been reported in corn (Jiang and Zhang 2002) and wheat (Luna et al. 2004). Moreover, Jung et al. (2006) referred to the role of catalase in the increase of drought tolerance in corn mutants for lessening the tension during the pollination stage.

3.3. The peroxidase activity

In the present study, the main effect of irrigation and inbred lines and the interaction effect between irrigation and inbred lines on the peroxidase activity was significant at 1% level(Table 1), showing 48.87% increase, with the highest growth(169.46%) in BGK 39 (60.63 mg g–1), under limited irrigation condition, in comparison to full irrigation condition (Table 2).The lowest peroxidase activity belonged in BGK 41 under full irrigation condition (12.02 mg g–1) (Table 3).

In a study, the stress of water shortage increased the activity of peroxidase in plant (Zhang et al. 2006). Generallyspeaking, the high activity level of antioxidant enzymes is associated with tolerance in the course of severe stress,such as drought or salinity (Sreenivasula et al. 2000; Srivalli et al. 2003; Guo et al. 2006).

Table 1 Analysis of variance for the effect of irrigation on physiological characteristics and yield in inbred lines of sunflower

Table 2 The mean comparison of irrigation effect on physiological characteristics and yield

Table 3 The mean comparison of irrigation effect on physiological characteristics in lines of sunflower

3.4. The polyphenol oxidase activity

The main effect of irrigation and interaction effect between irrigation and inbred lines also had a significant effect on the activity of polyphenol oxidase at 1% level (Table 1).In this experiment, 34.45% increase in the polyphenol oxidase activity was shown under limited irrigation condition compared to full irrigation condition (Table 2). Mean comparison results showed that the polyphenol oxidase activity in BGK 39 line was at the highest level under limited irrigation condition (63.43 mg g–1), which increased by 111.72% in comparison to full irrigation condition. The least activity of this enzyme was obtained at 22.75 and 22.57 mg g–1under limited irrigation condition in BGK 41 and under full irrigation condition in BGK 37, respectively (Table 4).

In Jaleel et al. (2007), the drought stress in the Catharanthus roseus plant increased catalase and polyphenol oxidase activities in comparison to control. In another experiment on the borage plant, the highest catalase and polyphenol oxidase activities were obtained under severe stress condition (Gholinegad et al. 2014).

It is believed that the increased activities of antioxidant enzymes are mainly responsible for resistance to environmental stresses in plants (Herbinger et al. 2002).

3.5. Protein content

According to the result of the analysis of variance, the main effect of irrigation and interaction effect between irrigation and line had a significant effect on protein at 1% level(Table 1). In this experiment, 14.98% increase of protein was seen under limited irrigation condition in comparison to full irrigation condition (Table 2). The results of the mean comparison of interaction effect showed that the highest protein content was related to BGK 41 under full irrigation condition (29.08). The amount of this characteristic in this line reduced to 20.43% under limited irrigation condition, in comparison to full irrigation condition. The lowest protein content was obtained in BGK 39 under limited irrigation condition (13.19 mg g–1) (Table 4). During drought, plants start to degrade proteins and accumulate some free amino acids to keep and adjust the cells’ osmotic pressure and reduce the synthesis of proteins (Moran et al. 1994).

3.6. Seed yield in each inbred line of sunflower

Our results revealed that the main effect of irrigation and interaction effect between irrigation and inbred lines had a significant effect on seed yield in all inbred lines at 1% level(Table 1). In this experiment, 45.33% increase in seed yield under limited irrigation condition was shown in comparison to full irrigation condition (Table 2). The highest seed yield was seen in BGK 1 and BGK 375 under full irrigation condition,at 45.8 and 44.9 g, respectively. Seed yield in BGK 1 andBGK 375 under limited irrigation condition decreased by 36.46 and 22.13%, respectively.

Table 4 The mean comparison of irrigation effect on polyphenol oxidase, seed yield, and protein in inbred lines of sunflower

RGK 23 produced the lowest protein content at 6.87 g in limited irrigation (Table 4). Water affects many physiological aspects of plant growth. Disorder in grain filling due to the increase in pollen infertility (Reddy et al. 2003) and damage to photosynthetic organs due to early defoliation (Rauf and Sadaqat 2008) are the main reasons for the decrease in yield under limited irrigation condition. In general, the growth of sunflower under limited irrigation condition depends on the balance between moisture status of plants and photosynthetic rate, and the proline content, osmoregulation,fluorescence, and chlorophyll index. The general growth of plants is a reflection of their tolerance and production stability under limited irrigation condition (Ghaffari et al. 2014).

According to Yegapan et al. (1996), drought stress in sunflower leads to early leaf senescence and a decrease in the number of leaves, leaf surface, 1 000-seed weight,and seed yield. A handful of other studies have verified the adverse effect of drought on seed yield (Ghaffari et al. 2014;Zabet et al. 2015).

3.7. Relative water content (RWC)

The results obtained from the experiment showed that the main factors of irrigation and inbred lines of sunflower have a statistically significant impact on the relative water content(Table 1). The RWC under limited irrigation condition decreased to 21.92% compared to full irrigation condition(Table 2). The highest and lowest RWCs in full irrigation condition were observed in BGK 357 (62.57%) and BGK 329(51.69%), respectively. The highest and lowest RWCs in limited irrigation condition were observed in BGK 375(51.80%) and BGK 329 (42.44%), respectively (Fig. 1).

Most studies have shown that when the leaf is exposed to drought, RWC, and water potential start to reduce (Li and Van Staden 1998; Decov et al. 2000; Nayyar and Gupta 2006). Drought reduces the relative humidity of leaves in sunflower, but the degree of reduction is lower in tolerant genotypes (Unyayar et al. 2004; Miyashita et al. 2005;Hossainet al. 2010).

4. Conclusion

According to our results, water withholding at the flowering stage led to a significant increase in proline content and in the catalase, peroxidase, and polyphenol oxidase activities.Limited irrigation also caused a significant decrease in protein content, seed yield in all inbred lines, and RWC.The responses of different lines of sunflower to antioxidant enzymes and proline and protein contents, and yield were significantly different in limited and full irrigation. The highest seed yields were obtained in BGK 1 and BGK 375 under full irrigation condition. The highest catalase, peroxidase,and polyphenol oxidase activities were observed in BGK 39 under limited irrigation condition with 114.35, 169.46,and 11.72% increase, respectively, in comparison to full irrigation condition, suggesting much greater adaptation to drought in this line. Antioxidants play a very obvious role in keeping the cell balance, because these metabolites are capable of direct reaction with reactive oxygen species and scavenge them.

Fig. 1 Relative water content (RWC) among different inbred lines of sunflower in full and limited irrigation conditions.

Acknowledgements

The authors are thankful to the Agricultural Research Station of Khoi, Iran for allowing me to use their land and to the University of Mohaghegh Ardabili, Iran for the use of laboratory facilities and materials.

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