Abstract
Melatonin, a substantial hormone, is a natural antioxidant agent that functions as a protector against the harmful effects of free radicals. Studies have found that “exogenous melatonin” applications have a positive effect on the growth and development of plants. This study investigated the adaptation of the seedlings that were transported from the nursery to the afforestation site for the process of planting. In 2019 the 2+0 aged bare-rooted Kastamonu/Taşköprü Anatolian Black pine seedlings, which are suitable for planting in semi-arid areas, were selected as research materials. Four different doses of “exogenous melatonin” (250, 500, 1000, and 1500 μM) were administered through two different methods (root-dipping and needle-spraying). Morphological seedling characteristics and bioactive chemical variables were measured for the control group and the seedlings treated with different doses of melatonin. Antioxidant enzyme activities were identified. When both the needle-spraying and root-dipping methods for melatonin application were evaluated in terms of morphological and biochemical variables, the best results were determined in low doses (250 to 500 μM). The results suggest that melatonin provides support to the metabolic process for the resistance of seedlings to low temperatures and semi-arid climatic conditions.
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Effects of Melatonin Applications on Anatolian Black Pine (Pinus nigra J. F. Arnold. subsp. pallasiana (Lamb.) Holmboe) Afforestation Performance in Semi-Arid Areas
Esra Nurten Yer Çelik,a,* Sezgin Ayan,a Halil Barış Özel,b Nezahat Turfan,c Batın Mehmet Yer,d and Gülbahar Abdaloğlu e
Melatonin, a substantial hormone, is a natural antioxidant agent that functions as a protector against the harmful effects of free radicals. Studies have found that “exogenous melatonin” applications have a positive effect on the growth and development of plants. This study investigated the adaptation of the seedlings that were transported from the nursery to the afforestation site for the process of planting. In 2019 the 2+0 aged bare-rooted Kastamonu/Taşköprü Anatolian Black pine seedlings, which are suitable for planting in semi-arid areas, were selected as research materials. Four different doses of “exogenous melatonin” (250, 500, 1000, and 1500 μM) were administered through two different methods (root-dipping and needle-spraying). Morphological seedling characteristics and bioactive chemical variables were measured for the control group and the seedlings treated with different doses of melatonin. Antioxidant enzyme activities were identified. When both the needle-spraying and root-dipping methods for melatonin application were evaluated in terms of morphological and biochemical variables, the best results were determined in low doses (250 to 500 μM). The results suggest that melatonin provides support to the metabolic process for the resistance of seedlings to low temperatures and semi-arid climatic conditions.
DOI: 10.15376/biores.18.2.2551-2572
Keywords: Exogenous melatonin; Pinus nigra subsp. pallasiana; Reactive oxygen species; Needle-spraying; Root-dipping
Contact information: a: Kastamonu University, Faculty of Forestry, Department of Silviculture, Kastamonu, Turkey; b: Bartın University, Faculty of Forestry, Department of Silviculture, Bartın, Turkey; c: Kastamonu University, Faculty of Science and Literature, Biology Department, Kastamonu, Turkey; d: Istanbul University-Cerrahpaşa, Faculty of Forestry, Department of Forest Yield and Biometry İstanbul, Turkey; e: Kastamonu Regional Directorate of Forestry, Kastamonu, Turkey;
* Corresponding author: esranurtenyer@gmail.com
GRAPHICAL ABSTRACT
INTRODUCTION
A number of adaptation challenges are seen with the lifting and transfer of bare-rooted seedlings raised in nursery conditions and planting them at afforestation sites. This challenge is more common in afforestation in arid, semi-arid, and anthropogenic steppe areas (Ayan et al. 2021). The adaptation of the seedling to the new environment of its planting is defined as acclimatization (Dirik 1990; Kijowska-Oberc et al. 2020; Yasmeen et al. 2022). The factors affecting planting success can be environmental and depend on the timely and intensive implementation of the applied technical processes. The rate of seedling viability is the direct result of the interaction of morphological, physiological, and genetic qualities that the seedling has. In particular, root damage during the lifting of bare-rooted seedlings on the nursery bed and planting them at the afforestation site negatively affect the root-to-shoot ratio (Harris 1992; Ledo et al. 2018).
Special operations have been implemented to enhance planting success, particularly on arid and semi-arid lands. Preservatives applied to the root region of the seedling prior to planting are one of these applications. “Exogenous melatonin” applications have been reported to have a positive effect on plant growth and development and the acquisition of tolerance to abiotic stress conditions (Li et al. 2012; Turk et al. 2014; Bajwa et al. 2014; Wei et al. 2015; Lee and Back 2017; Nawaz et al. 2018; Shen et al. 2021). Melatonin (N-acetyl-5-methoxytryptamine), a natural antioxidant, is an important hormone that performs the function of protecting biological tissues from the harmful effects of free radicals. Melatonin is known to be a preserved molecule in the evolutionary process in all species of living things and was first discovered in plants in 1995 (Van Tassel et al. 1995, 2001). Initial research in plants focused on Chenopodium rubrum (lamb’s quarters), Eichhornia crassipes (water hyacinth), Vitis vinifera (grape vine), Prunus avium (cherry), and Ulva sp. (green algae) (Wolf et al. 2001; Tan et al. 2007; Boccalandro et al. 2011; Tal et al. 2011; Zhao et al. 2013).
Melatonin functionally delays leaf abscission in plants. It is tasked with the role of growth regulator, such that it enables the development of tissues such as root and shoot. It was also determined to be a natural stimulus against various stressors (Shen et al. 2021; Zhao et al. 2021). It has been shown by researchers that it is an important signal molecule against abiotic stresses such as drought, salt, cold, frost, temperature, etc. It is a critically important antioxidant molecule that is tasked with rapidly scavenging free radicals formed at the cellular level by stress (Galano et al. 2011; Arnao 2014; Bajwa et al. 2014; Zhang et al. 2014; Arnao and Hernandez-Ruiz 2015; Jiang et al. 2016; Chen et al. 2018; Kanwar et al. 2018; Shen et al. 2021). Furthermore, the application of “exogenous melatonin” against oxidative stress regulates the plant defense mechanism (Tan et al. 2012; Wang et al. 2015; Zhang et al. 2017). Melatonin neutralizes hydroxyl (OH–⋅), superoxide (O2–⋅), and NO–⋅ radicals, all of which show high impact. It increases the activity of various antioxidants such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and guaiacol peroxidase (GPx) enzymes (Baydaş et al. 2001). Melatonin functions as an antioxidant by scavenging free radicals, stimulating the activity of antioxidant enzymes, reducing electrical conductivity, and increasing mitochondrial oxidative phosphorylation (OXPHOS) (Reiter et al. 2003). Externally applied melatonin inhibits drought stress by increasing the activity of antioxidant enzymes (Wang et al. 2013; Li et al. 2015). Li et al. (2016) determined that the external application of melatonin of 0.05 mmol/L to canola (Brassica ssp.) seeds increased the content of soluble sugar and protein and prevented growth decline caused by drought stress. Cao et al. (2019) investigated the effectiveness of the melatonin hormone against frost harm in harvested peach berry. Frost harm was decreased in melatonin-treated fruits. Melatonin stimulates the contents of the hydrogen peroxide (H2O2) at the first stage but inhibits it in the subsequent process; it increases the expression of genes responsible for antioxidants. In brief, melatonin increases synthesis genes, reduces oxidative stress, restores redox balance, and improves the activity of antioxidant enzymes (Xin et al. 2017; Wei et al. 2018; Gholami and Zahedi 2019; Gholami et al. 2022).
Seedlings grown in open-field nursery conditions experience certain levels of stress during the transplant and planting stages to afforestation sites, failing to demonstrate the desired growth performance. With the aim of increasing the success of seedlings at plantation sites and preventing the stress experienced by seedlings during the planting process, this study investigated whether external melatonin applied to the seedling will be successful with hormone use. The effect of melatonin applications on afforestation performance of bare-rooted 2+0 aged Anatolian Black pine seedlings (Pinus nigra J. F. Arnold. subsp. pallasiana (Lamb.) Holmboe) was determined.
EXPERIMENTAL
This study was conducted using 2+0 aged bare-rooted provenances of Kastamonu/Taşköprü Anatolian black pine in a 76.8 ha plantation site in Karasapaca Village (Latitude: 41048626; Longitude: 3407123) within the borders of Kastamonu-Tosya Forest Enterprise Directorate between 2018 and 2020 in Turkey. The area has an average altitude of 910 m a.s.l. and the annual rainfall amount is 467 mm. The study area is south-facing, with a slope ranging from 31 to 60%. The soil structure is sandy-clay and its bedrock has a sediment rock structure.
Anatolian black pine seedlings were grouped as to four different doses (250 μM – 500 μM – 1000 μM – 1500 μM) and Control group (Wang et al. 2013) and planted in the site on 07 March 2019. Two different applications forms (root-dipping / needle-spraying) were used (Yer Celik 2021). Before planting, the plants’ roots were soaked in melatonin for 30 minutes (root-dipping). In the second application method, melatonin was applied to the needles of the seedlings by spraying every month during the vegetation period. Biochemical measurements were made using the seedlings three times during the vegetation period (beginning (April), mid (July), and end (November) of the vegetation period). As of the end of vegetation, morphological measurements were conducted on 30 seedlings in total (10 seedlings x 3 replications).
As morphological measurements, root collar diameter (RCD) was measured with a 0.1 mm precision digital caliper; seedling height (SH) was measured with a steel tape; The fresh weight of the shoot (FWS), the fresh weight of the root (FWR), and the total fresh weight of the seedlings (FWSD) were measured with a precision balance of 0.001 g. After drying, the values for the dry weight of the shoot (DWSH), the dry weight of the root (DWR) and the total dry weight of the seedlings (DWS) and number of buds (NB), number of branches per seedling (NBS), and root length (RL) parameters were measured. Besides, the following values were calculated from these obtained values,
where %Root, SI, LI and DQI represent the root percentage, sturdiness index (Aphalo and Rikala 2003), layering index and Dickson quality index (Ayan 2002).
As biochemical variables, H2O2, MDA, chlorophyll, proline, protein, sugars, as well as, antioxidant enzyme activities such as SOD and POD were investigated. The detection of proline amount was made according to the method of Bates et al. (1973), protein content according to Bradford (1976) method, malondialdehyde (MDA) content according to Lutts et al. (1996), and hydrogen peroxide (H2O2) according to Velikova et al. (2000). The method of Witham et al. (1971) was used to determine photosynthetic pigments. SOD activity was measured according to nitro blue tetrazolium chloride (NBT) reduced by O2– under light (Agarwal and Pandey 2004). POD activity was determined according to Yee et al. (2002). All analyses were performed with three replications. Data of morphological and biochemical variables obtained as a result of the measurements were subjected to variance analysis (ANOVA). The Duncan test was used for intergroup binary comparisons when statistically significant differences (p<0.05) were found in terms of measured values. Statistical analyses were done using IBM SPSS Statistics 22 package software.
RESULTS
Effect of Melatonin Applications at Different Doses on Morphological Characters
The effects of exogenous melatonin, applied at different doses to different parts of the plants, on root collar diameter, number of branches per seedling, number of buds, root length, fresh weight of the shoot, dry fresh weight, the total fresh weight of the seedlings, the dry weight of the shoot, and sturdiness index values of the seedlings were found to be significant according to the variance analysis results (p<0.05). Variance analysis of morphological characters and results of the Duncan’s Test are presented in Table 1. The highest values in terms of RCD and RL compared to the control were determined in the 500 μM melatonin treatment applied to seedling roots before planting. The highest NBS compared to control was detected in seedlings at the treatment doses of 250 and 500 μM sprayed on seedling shoots. According to the statistical values of the number of buds in the seedlings at the end of vegetation, the highest NB was recorded in the seedlings treated with 250 μM melatonin to root zone. In terms of FWS, FWR, and FWSD values; the highest values compared to the control group were detected in the dose of 500 μM melatonin spraying, respectively. The highest DWSH was achieved in the 250 μM dose of melatonin applied by needle-spraying. In terms of its effect on morphological seedling parameters, low doses of melatonin (250 μM, 500 μM) yielded more positive results in both the needle-spraying and root-dipping.
Effect of Different Doses of Melatonin Application on Bioactive Chemical Components
Its effect on nitrogen compounds (proline, total soluble protein)
The variation of proline quantity because of applying melatonin to plant parts at different doses through different ways of application is given in Table 2. The dose, time, and way of application were determined to cause statistically significant variations (p<0.05) on the studied parameters. Analyses conducted at the beginning of the vegetation period (April), mid-vegetation period (July), and at the end of the vegetation period (November) found that proline contents increased in all treatments compared to the control group in April and July.
Table 1. Statistical Values of Sapling Morphological Characters
According to all melatonin applications in November, higher proline was determined in control seedlings. In April, while vegetation was in its beginning, the melatonin dose of 1,500 μM applied to seedling roots constituted a higher proline value compared to control and all other treatments. In the mid-vegetative month of July, the application of melatonin in the form of spraying increased the proline value, also the highest proline value was ensured with the 500, 1000, and 1500 μM doses applied with the needle-spraying to shoots.
In terms of protein values (Table 2), the highest values were determined in the 250 μM treatment applied to the root part of the seedling in April and the 500 μM melatonin treatment applied to the seedling shoots in July. The control seedlings had a higher protein value in all treatment doses and administration ways in November after the vegetation period was completed.
Table 2. The Effect of Melatonin Applications at Different Doses on Nitrogenous Compounds
Its effect on enzyme activities (superoxide dismutases/SOD and peroxidase/POD)
When studying the effects of melatonin on antioxidant enzyme activities, no significant differences were detected in April SOD activity (Table 3). In July, SOD activity showed high value in all melatonin applications compared to control. The application of 250 μM melatonin, specifically by needle-spraying, showed the highest enzyme activity (16.61 EU). A significant decrease in SOD was detected in November compared to July (Table 3). As for POD activity, the highest POD values were determined in the Control treatment in November, 1000 μM needle-spraying in April, and 500 μM in July (Table 3).
Table 3. Effect of Melatonin Applications at Different Doses on Enzyme Activities
Its effect on carbon compounds (glucose, sucrose)
The highest values of sucrose were determined in all of the months of April, July, and November, following the application at the dose of 250 μM to the root. On the other hand, it was determined that different treatment combinations in different periods created the highest glucose content value.
The highest glucose content was determined by the spraying of 1000 μM melatonin to the needle leaves in April, while 250 μM melatonin application to the roots and needle leaves in July produced the highest glucose value, and in November, the highest dose was achieved by 1500 μM melatonin treatment applied to the root within the scope of the research (Table 4).
Its effect on oxidative stress (H2O2, MDA)
When the effects of different doses of melatonin applications on the amount of H2O2 were examined, the H2O2 concentration was found to be higher in all melatonin treatment groups compared to the control in both April and July. On the other hand, a higher H2O2 value was detected in the control treatment in the dormant phase of the seedlings in November compared to all melatonin treatment combinations (Table 5). In April, the H2O2 content was the highest in seedlings that were treated with both needle-spraying and root-dipping at the melatonin dose of 500 μM.
In July, H2O2 was at the highest level at 500 μM melatonin applications through needle-spraying and 250 μM through root-dipping (Table 5). The MDA concentration was the highest in the control in April, while it was detected that the spray application of melatonin at the dose of 250 μM to the needle leaves in July and November caused the highest concentration (Table 5).
Table 4. Effect of Melatonin Application at Different Doses on Carbonated Compounds
Table 5. Effect of Melatonin Applications at Different Doses on Oxidative Stress