Abstract: Background and Objective: The adverse effects of drought can be mitigated by application of natural plant extracts as carrot root or antioxidants as β-carotene. Carrot root is characterized by high nutritional value and rich with bioactive constituents and antioxidants (phenolic compounds and carotenoids). This work aimed to compare between the physiological role of carrot root extract (5 and 10%) and β-carotene (25 and 50 mM) as seed soaking in inducing drought tolerance of sunflower plant (Giza 102 cultivar). Materials and Methods: Two pot experiments were conducted during two successive summer seasons (2015 and 2016) at wire house of National Research Centre, Egypt. The sterilized seeds were divided into three groups, the first group was soaked with distilled water, while second and third groups were soaked for 12 h with different concentrations of carrot root extract (5 and 10%) and β-carotene (25 and 50 mM). Results: Data showed that drought stress (50% FC) decreased quality and quantity of sunflower plants. Meanwhile carrot root extract and β-carotene mitigate the adverse effects of drought and induce plant tolerance through enhancement plant growth regulators; photosynthetic pigments; antioxidant enzymes; IAA; phenolic compound; osmolytes (TSS; proline, free amino acids); seed yield and seed composition as protein and oil content. Conclusion: Carrot root extract treatments were more pronounced than β-carotene treatments. Worthy, 10% carrot root extract was the most pronounced treatment in increasing drought tolerance of sunflower plant.
INTRODUCTION
One of the seriously abiotic stresses that negatively affect production of world agriculture by over 25% is the continuous or frequent drought1 that affects about 45% of the world agricultural2. Drought induces different morphological, physiological, biochemical and molecular changes in plants3,4 and exerts negative effects on cellular membranes and organelles such as mitochondria and chloroplasts5 and causing cellular content leakage outside the cell6 thereby losses in tissue water content which reduce turgor pressure in the cell and inhibiting enlargement and division of cell leading to reduction in plant growth and dry mass accumulation7. Moreover, drought significantly reduced the activities of enzymes responsible for some processes like respiration, photosynthesis, translocation, hormonal balance, uptake of macro and micro nutrients and their metabolism8. Apel and Hirt9 stated that the secondary negative effect of drought increased the generation of ‘reactive oxygen species’ (ROS), including highly reactive free radicals such as singlet oxygen, superoxide, hydroxyl radicals, hydrogen peroxide and other strong oxidant molecules. So under environmental stress, plants activate antioxidant systems, either enzymatic (e.g., peroxidase,, superoxide dismutase, catalase, glutathione reductase) or non-enzymatic (e.g., vitamins E and C, carotenoids, flavonoids and other phenolic compounds etc.) as reported by Apel and Hirt9. On the other hand, plants tend to accumulate some osmoregulators such as soluble sugars, glycinebetaine, proline, free amino acids, mannitol, phenolics etc. In addition, under dehydration conditions, these osmolytes act as osmoprotective substances via protecting cells from oxidative stress by scavenging ROS1,10.
The adverse effects of different abiotic stresses can be mitigated by application of natural plant extracts as carrot root11 or antioxidants as β-carotene.
Carrot (Daucus carota L.) is characterized by high nutritional value and bioactive constituents12. It is rich with antioxidants of hydrophilic (phenolic compounds) and lipophilic (carotenoids) nature12,13. It is worthy to mention that flavonoids and phenolic derivates present in carrot roots may play an important role as antioxidants as mentioned by Zhang and Hamauzu14. Carrot root extracts included detoxifying agents called indoles as well as amino acids, sugar, proteins and fibers15 and showed growth-stimulating activities and used as biostimulants in promoting plant growth and crop production16,17. Puchooa and Ramburn18 showed that with increasing concentration of carrot juice in the medium, the fresh weight, dry weight and moisture content of the explants were increased. Abbas and Akladious11 mentioned that carrot root extract increased growth parameters, photosynthetic pigments and total carbohydrate content as well as antioxidant substances (ascorbic acid, anthocyanins, phenolic compounds and flavonoids) of the cowpea seedlings either under normal or salinity stressed conditions where 25 mg L–1 carrot root extract was more effective in reducing the harmful effects of salinity by the enhancement of multiple processes. Abou El-Ghit19 indicated that extract of carrot seeds showed positive allelopathic effects, improved seed germination, growth and metabolic activities as increased auxin content and decreased abscisic acid content of pea seedlings. Likewise, Recently, Kasim et al.20 recommend that priming Vicia faba seeds with carrot extract as a new natural and low-cost method not only for the alleviation of drought stress on the plants but also for stimulating their growth.
β-carotene is responsible for giving carrots their orange color and exerts different functions in plants, besides its direct role in photosynthesis, it is involved in the mechanisms of oxidative stress tolerance21. The main physiological function of β-carotene is the precursor of vitamin A (retinol)22. β-carotene acts as a potent antioxidants via deactivation of free radicals and singlet oxygen quenching23-25. Carrot is one of the best sources of β-carotene. The carotene content of carrots ranges from 60-120 mg/100 g but some varieties can contain26 up to 300 mg/100 g.
Sunflower (Helianthus annuus L.) could be cultivated in different types of soils and climate conditions27. Sunflower oil is quite palatable and considered as an important source of edible vegetable oil.
This work aimed to investigate which is more effective carrot root extract or β-carotene treatments in ameliorating the deleterious impact of severe drought stress on the performance of sunflower plant.
MATERIALS AND METHODS
Preparation of carrot root extract: Extraction of carrot root was done according to Sofowora28. Sample of fresh orange carrot root was collected from the local markets in Egypt. About 100 g of washed carrot root was sliced into tiny pieces and blended in an electric blender with 160 mL distilled water and 160 mL of 80% ethanol and shake for 1 h. Then, the extract was filtered with Whatman No. 1 filter paper and the filtrate was adjusted to pH 7.0 with 1 N NaOH and completed with distilled water to 1 L. The collected extract was used to make 5 and 10% carrot root extract.
Pot experimental design: Two pot experiments were conducted during two successive summer seasons (2015 and 2016) at wire house of National Research Centre, Egypt. Sunflower seeds (Giza 102 cultivar) were obtained from Agricultural Research Centre, Giza, Egypt. The seeds were sterilized with 2% sodium hypochlorite for 5 min and then washed several times with distilled water. The sterilized seeds were divided into three groups, the first group was soaked with distilled water, while second and third groups were soaked for 12 h with different concentrations of carrot root extract (5 and 10%) and β-carotene (25 and 50 mM). Then the seeds were air dried and sown along a centre row in each pot.
Regarding chemical fertilizers, Ca-superphosphate (15.5% P2O5) was applied at a rate of 10 g/pot before sowing. Nitrogen fertilizer as ammonium sulfate (20.5% N) was applied at the rate of 2 g/pot twice at 3 and 5 weeks old plants. Each experiment was carried out in plastic pots filled with equal amounts (3:1 w/w) of sieved clay/sandy soil.
The sunflower seeds were sown 2 cm deep during first of June. Each experiment comprised 10 treatments with 6 replicates in a complete randomized block design.
After two weeks from sowing, thinning was done leaving 2 seedlings per pot. The plants were watered regularly to Field Capacity (FC) till the drought treatments were imposed. The plants were exposed to drought stress after 21 days from sowing. The sunflower plants were subjected to drought stress (50% FC) while control plants were irrigated at 95% FC (full field capacity).
Samples of sunflower plant were collected at 60 days old to determine some growth parameters (shoot height, leaves number, fresh and dry weight of plant) photosynthetic pigments, indole acetic acid, phenolic contents, total soluble carbohydrates, proline and total free amino acids as well as some antioxidant enzymes (catalase, peroxidase, polyphenol oxidase and phenylalanine ammonia lyase) in the leaves.
At harvest, sunflower plants were collected to determine head diameter. Heads were air dried and threshed to determine seeds weight/plant and 100 seeds weight.
Chemical analysis of sunflower leaves: Photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in the fresh sunflower leaf were determined as the method described by Moran29. Indole acetic acid content was determined according to the method described by Larsen et al.30. Total phenolic compounds was determined according to the method described by Danil and George31 using Folin Ciocalteu reagent. Total Soluble Sugars (TSS) were extracted by overnight submersion of dry tissue in 10 mL of 80% (v/v) ethanol at 25°C with periodic shaking and centrifuged at 600 g. The supernatant was evaporated till completely dried then dissolved in a known volume of distilled water to be ready for determination of soluble carbohydrates according to Yemm and Willis32. Proline was estimated according to Bates et al.33. Total free amino acids were determined according to Muting and Kaiser34. The oil content of the yielded seeds was determined according to the procedure reported by AOAC35. The defatted meals were used for determination of the protein content by micro kjeldahl method according to AOAC35.
Assay of enzymes activities: Enzyme extracts were collected following the method described by Chen and Wang36. Leaf tissues were homogenized in ice-cold phosphate buffer (50 mM, pH 7.8), followed by centrifugation at 8,000 rpm and 4°C for 15 min. The supernatant was used immediately to determine the activities of enzymes. Peroxidase activity (POX) (EC 1.11.1.7) was assayed by the method of Kumar and Khan37. Polyphenol-oxidase (PPO, EC 1.10.3.1) was assayed using the method of Kar and Mishra38. Catalase activity (CAT) (EC 1.11.1.6) activity was determined spectrophotometrically by following the decrease in absorbance36 at 240 nm. The enzyme activities were calculated by Kong et al.39. The PAL activity was determined based on the rate of cinnamic acid production as described by Ochoa-Alejo and Gomez-Peralta40.
Statistical analysis: The averages of two growing seasons were statistically analyzed as a randomized complete block design system according to Gomez and Gomez41. The Duncan multiple range test was used to compare the treatment means at 5% levels of probability.
RESULTS
Vegetative growth parameters: Drought stress (50% FC) caused marked decreases in vegetative growth parameter of sunflower (shoot height, leaves number, fresh and dry weight of plant) relative to control plants (Table 1). On the other hand, carrot root extract treatments and β-carotene treatments caused marked increase in vegetative growth parameters of unstressed plants and drought stressed plants relative to corresponding controls. The positive effect of both carrot root extract and β-carotene was increased by increasing its dose. It was noted that carrot root extract treatments were more pronounced than β-carotene treatments. Where, 10% carrot root extract caused the highest increase in all vegetative parameters under investigation followed by 5% carrot root extract, while, 25 mM β-carotene showed the least increase either in unstressed plant or drought stressed plants.
| Table 1: | Response of vegetative growth parameters of sunflower plant grown under drought to carrot root extract or β-carotene |
| Means followed by the same letter for each tested parameter are not significantly different by Duncan’s test (p<0.05) | |
| Table 2: | Response of photosynthetic pigments of sunflower plant grown under drought to carrot root extract or β-carotene |
| Means followed by the same letter for each tested parameter are not significantly different by Duncan’s test (p<0.05) | |
Photosynthetic pigments: Drought stress significantly decreased all components of photosynthetic pigments relative to control (Table 2). Since, total photosynthetic pigment was decreased by 14.72% than control. On the other hand, all applied treatments caused significant increases in all components of photosynthetic pigments in unstressed plants or drought stressed plants relative to corresponding controls. The highest significant increase in total photosynthetic pigments was achieved by 10% carrot root extract followed by 50 mM β-carotene. Regarding unstressed plant (95% FC), 10% carrot root extract significantly increased total photosynthetic pigments by 28.63% and 50 mM β-carotene increased total photosynthetic pigments by 14.41%. Meanwhile, total photosynthetic pigments in drought stressed plants (50% FC) were increased by 10% carrot root extract by 26.01 and 20.36% due to 50 mM β-carotene relative to corresponding controls.
Antioxidant enzymes: Activity of antioxidant enzymes (peroxidase, polyphenol oxidase, catalase and phenylalanine ammonia lyase) were significantly decreased by drought stress (Table 3). Meanwhile, all applied treatments significantly increased activity of antioxidant enzymes in unstressed plants and drought stressed plants relative to corresponding controls.
| Table 3: | Response of antioxidant enzymes of sunflower plant grown under drought to carrot root extract or β-carotene |
| Means followed by the same letter for each tested parameter are not significantly different by Duncan’s test (p<0.05) | |
Regarding unstressed plants (95% FC), the highest increases in peroxidase and catalase activity were achieved by 10% carrot root extract followed by 50 mM β-carotene. Meanwhile, the highest increases in polyphenol oxidase and phenylalanine ammonia lyase activity were achieved by 10% carrot root extract followed by 5% carrot root extract. Worthily, 10% carrot root extract was the most pronounced treatment in increasing activity of antioxidant enzymes of drought stressed plants.
IAA and phenolic compound: Drought stress significantly decreased IAA by 20.54% relative to control (Table 4). Both carrot root extract and β-carotene treatments significantly increased IAA in unstressed plants and drought stressed plants. It is clear that carrot root extract treatments were more pronounced than β-carotene treatments. Where, 10% carrot root extract increased IAA in unstressed plants by 35.01% and in drought stressed plants by 63.46% relative to corresponding controls.
Regarding phenolic content, it was noted that drought stress significantly increased phenolic content by 17.27% relative to control (Table 4). All applied treatments significantly increased phenolic content. Carrot root extract treatments were more pronounced than β-carotene treatments. About 10% carrot root extract was more effective than 5% carrot root extract.
Total soluble sugars (TSS), proline and free amino acids: The TSS was significantly decreased by drought stress by 9.09% relative to control (Table 4). Meanwhile, all applied treatments significantly increased TSS in unstressed plants or drought stressed plants. About 10% carrot root extract was the most pronounced treatment followed by 5% carrot root extract.
Table 4 showed that drought stress significantly increased proline and free amino acids by 17.26 and 8.11% relative to control. Whereas, carrot root extract and β-carotene treatments significantly increased proline and free amino acids under all conditions. About 10% was the most pronounced treatment.
Seed yield, its components and seed chemical composition: Drought stress significantly decreased seed yield by 21.13% relative to control (Table 5). On the other hand, all applied treatments significantly increased seed yield and its components in unstressed plant and drought stressed plants relative to corresponding control. Worthily, 10% carrot root extract was the most pronounced treatments, since, it increased seed yield by 49.63% in unstressed plants and by 48.83% in drought stressed plants relative to corresponding controls.
Regarding chemical composition of the yielded seeds, Table 5 showed that drought stress decreased protein content by 17.14% and oil content by 11.43% relative to control plants. All applied treatments significantly increased both protein and oil content of the yielded seeds in unstressed plants and drought stressed plants relative to corresponding controls. The highest significant increases in protein and oil content in unstressed plants was achieved by 10% carrot root extract followed by 50 mM β-carotene relative to unstressed control. Whereas, drought stressed plants showed significant increases in protein and oil content due to carrot root extract treatments.
| Table 4: | Response of IAA, phenolic compound and some osmolytes of sunflower plant grown under drought to carrot root extract or β-carotene |
| Means followed by the same letter for each tested parameter are not significantly different by Duncan’s test (p<0.05) | |
| Table 5: | Response of seed yield, its components, protein and oil content of sunflower plant grown under drought to carrot root extract or β-carotene |
| Means followed by the same letter for each tested parameter are not significantly different by Duncan’s test (p<0.05) | |
DISCUSSION
Drought stress reduced the plant growth (Table 1) might be due to the metabolic disorders induced by stress; generation of ROS; reduction in cell division and elongation, cell turgor and eventually cell growth42-44.
Table 1 showed that carrot root extracts and β-carotene treatments stimulated sunflower vegetative growth parameters. These increases may be attributed to the components of carrot root extract that have defense action against abiotic stress and influenced plant growth by regulating many physiological processes as mentioned by Sairam et al.45. In addition, Doco et al.46 attributed the increase in cowpea seedlings growth treated with carrot extract to the presence of the phytohormone. Likewise, the increase in fresh weight, dry weight and moisture content of the explants of Daucus carota with increasing carrot juice concentration in the medium was reported by Puchooa and Ramburn18. β-carotene acts as a potent antioxidants via deactivation of free radicals and singlet oxygen quenching23-25 and involved in the mechanisms of oxidative stress tolerance21.
The reduction of photosynthetic pigments under drought stress (Table 2) may be attributed to the oxidation of chloroplast lipids and changes in the structure of pigments and proteins47 or due to chlorophyll degradation by the formation of proteolytic enzymes like chlorophyllase, deterioration in chloroplast and stomatal closure48.
Carrot root extract significantly enhanced photosynthetic pigments (Table 2). These increases may be due to the reduction in chlorophyll degradation and activation of enzymes that regulate photosynthetic carbon reduction and protect chloroplast from oxidative damage49. Moreover, carrot root extract is rich with macro and micronutrients such as N, P, K, Mg, Fe and Cu, which enhancing the formation of chlorophyll50,51.
β-carotene is essential in photosynthesis where it acts as energy carriers and photo-oxidation protectors because carotenoids are free radical scavengers. These increases in photosynthetic pigments due to carrot root extracts and β-carotene treatments may be attributed to the role of carotenoids in protecting chlorophylls from photo-oxidative destruction as mentioned by Devlin and Withman52 and Abbas and Akladious11.
Data recorded in Table 3 indicated that peroxidase, polyphenol oxidase, catalase and phenylalanine ammonia lyase activities were decreased in sunflower leaf under drought stress. These results are in agreement with those achieved by Maria et al.53 who revealed that there was a great decrease in the activity of phenoloxidase in the wheat leaves with increasing the water deficit. Iqbal54 reported that drought stress decreased peroxidase activity in wheat leaves. Abedi and Pakniyat55 attributed the decline in POD activity to the inhibition of enzyme synthesis or change in the assembly of enzyme subunits under stress conditions.
On the other hand, all applied treatments increased the activity of antioxidant enzymes under investigation in sunflower plant irrigated with either enough quantity of water or undergo drought stress (Table 3). Kasim et al.20 stated that carrot root extract contains vitamin A (as β-carotene) which may act as ROS scavengers resulting in improvement of plant growth. Carotenoids play a major role in the protection of plants against photo-oxidative processes. They are efficient antioxidants scavenging singlet molecular oxygen and peroxyl radicals56.
β-carotene exhibits antioxidant properties57. Due to their ability to dissolve in fat, carotenoids affect many biological processes, including photosynthesis or sweeping of free radicals and singlet oxygen58. High activity of CAT indicated drought tolerance in some of the canola cultivars59. Abedi and Pakniyat55 showed that SOD and POX activities were increased in some canola cultivars and linked with protection from oxidative damage due to drought stress. Gharache et al.60 mentioned that increases in antioxidant enzyme activities promote the removal of reactive oxygen species (ROS), thus enhancing plant drought resistance. The increase in CAT and POD activities may help in a better protection mechanism against oxidative damage under water stress61.
Drought significantly decreased IAA (Table 4). Abiotic stress may be caused some modification in the production of phytohormones which is influenced by changes of enzyme activity that participates in phytohormones synthesis or /and degradation62. On the other hand, all applied treatments increased IAA relative to corresponding controls. Since, Spectrophotometric and chromatographic analyses of the carrot juice, revealed the presence of indole-3-acetic acid (IAA) as reported by Puchooa and Ramburn18.
Drought and all applied treatments significantly increased phenolic content (Table 4). The level of non-enzymatic antioxidant as phenolic compounds under abiotic stress was increased, due to their capacity to protect itself against oxidative stress. They directly react with super-oxide anions and lipid peroxyl radicals that consequently reduce or breakdown the chain of lipid peroxidation63.
Regarding effect of carrot root extract and β-carotene treatments, Carrots extract contain bioactive compounds, such as phenolic compound and carotenoids that act as scavenger of free radicals64. Phenolic compounds in carrot root extract play an important role in the biosynthesis process of non-enzymatic antioxidants in response to various environmental stresses and stimulate the plant growth and development due to their antioxidant capacity65 and increases plant resistance to undesirable effects of biotic and abiotic stresses. In vitro, inclusion of carrot juice in growth medium of Daucus carota increased the level of antioxidants as phenolic compounds66.
Table 3 showed that drought stress decreased TSS and increased proline and free amino acids. Meanwhile all applied treatments increased TSS, proline and free amino acids. The reduction in TSS due to drought stress could ascribe to loss of solutes from guard cells, which resulted in a selective reduction in guard cells turgor leading to stomatal closure. Zhang et al.67 mentioned that the soluble carbohydrate concentration in well-watered wheat plants was higher than those of stressed plants.
Proline is generally considered as a good indicator of abiotic stress in different species68-70. Proline not only acts as an osmolyte but also contributes in stabilizing subcellular structures (e.g., membranes and proteins), scavenging free radicals and buffering cellular redox potential under stress conditions54 and serves as nitrogen storage or osmoregulator solute that help plant to tolerate stress condition71. Abass and Mohamed72 showed that the drought condition caused significant increase in the proline content in shoot of common bean (Phaseolus vulgaris L.) plants.
The role of amino acids in abiotic stress tolerance was reported by Singh73, since; these molecules are thought to play a pivotal role in plant cytoplasmic osmotic adjustment in response to osmotic stress.
On the other hand, all applied treatments significantly enhanced TSS, proline and free amino acids (as osmolytes). These enhancements may be attributed to the role of carrot root extract and β-carotene as strong antioxidants thereby scavenge ROS that formed within plants under normal condition or stressed condition and increased drought tolerance of plant via osmotic adjustments.
It is apparent that seed yield and yield components were reduced due to drought compared to control (Table 5). This reduction was due to reduction in water and nutrient uptake and transport in plant which affects rate of translocation of photosynthates to seed filling.
Drought stress reduced the crop yield due to reduction in photosynthetic pigments74 and diminished activities of calvin cycle enzymes75. Ali et al.76 stated that changes in seed chemical composition could have been due to the reason that low water supply during the plant life affects many enzymes whose activity is reduced under water stress conditions and leading to changes in metabolic activities that result in altered translocation of assimilates to seeds. Ali and Alqurainy77 mentioned that the main cellular components susceptible to damage by free radicals are lipids (peroxidation of unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids. The reduction in the oil content under drought stress could be due to oxidation of some of the polyunsaturated fatty acids78. Generally, drought stress reduces growth79 and yield of various crops80 by decreasing chlorophyll pigments, photosynthetic rate, stomatal conductance and transpiration rates81.
Carrot root extract contains ascorbic acid, auxin substances (IAA), gibberellic acids, kinetin and benzyl adenine as cytokinins, K, P, Mg and Zn as reported by Kasim et al.20. These ingredients are known to improve the growth and increase cell division, cell enlargement and translocation of assimilates from source to sink82-84 and reflected on seed yield and its components. Carrot roots extract contains auxins and cytokinins which have beneficial effect on carbohydrates accumulation16.
Regarding to oil and protein contents of the yielded seeds, it was noted that drought stress decreased the both parameters, while carrot root extract and carotene increased them. Water deficit leads to a fall in the content of the proteins as well as modifying their composition85. Ozturk et al.86 found that protein content was significantly decreased, while proline was accumulated with increasing in salinity level. The reduction in the oil content under drought stress could be due to oxidation of some of the polyunsaturated fatty acids78.
CONCLUSION
Carrot root extract treatments were more pronounced than β-carotene treatments. Worthy, 10% carrot root extract was the most pronounced treatment in increasing drought tolerance of sunflower plant.