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. 2023 Feb 3;29(2):185–193. doi: 10.1007/s12298-023-01284-x

The application of methyl jasmonate in combination with ascorbic acid on morphological traits and some biochemical parameters in red willow

Fatemeh Sahraei 1, Mousa Solgi 1,✉, Mina Taghizadeh 1
PMCID: PMC9981849  PMID: 36875731

Abstract

Red willow, an economically important ornamental shrub in Iran, is characterized mainly by its red stems, making it a valuable ornamental plant in flower markets. This study aimed to investigate the effect of foliar application of methyl jasmonate (MeJA) and ascorbic acid on the morphological and biochemical characteristics of red willow. The experiment was conducted as a completely randomized design with two factors and three replications. Juvenile willow shrubs (3 to 4-year-old red) were cultivated in Hossein Abad village, Markazi province, Iran. The experimental treatments consisted of MeJA (0, 100 and 200 mgL−1) and ascorbic acid (0, 100 and 200 mgL−1). Several traits were evaluated such as the longest branch and two nearest heights, total shrub diameter, the longest branch diameter (at lower, middle and upper parts), total anthocyanin content of the longest branch, salicin content, leaf chlorophyll (a, b and a + b) content, and carotenoid content. In addition, the number, length and width of leaves from the longest branch, fresh and dry weight of branches were analyzed. Results revealed that the application of MeJA and ascorbic acid significantly increased growth characteristics (height, leaf number, total shrub diameter, branch diameter, fresh and dry weight and total anthocyanin content) of red willow shrubs. Furthermore, it was found that 200 mgL−1 treatments of these two substances produced the best results. Red willow shrub growth parameters and yield were also improved by the interaction of these two factors. Additionally, a significant correlation was found between total anthocyanin content and leaf number of the longest branch, total shrub diameter, the height of nearest branch 2 and the plant fresh weight.

Keywords: Anthocyanin, Ascorbic acid, Methyl jasmonate, Red salix, Salicin

Introduction

Red Salix or Red Willow (Salix purpurea L.) is a small, deciduous ornamental shrub with dense, narrow branches that is green in the warm seasons and purple to red in the cold seasons. This plant is of great interest to growers because of its red color and special attractiveness (Isebrands and Richardson 2014), which contributes significantly to its marketability (Solgi and Sahraei 2022). Moreover, it provides society with benefits by rehabilitation degraded land, reducing the climate change impact, and restoring forest landscapes (Xu et al. 2021, 2009; Kuzovkina and Volk 2009). Therefore, ensuring the right conditions for proper growth and proper branch coloring can help to successfully capture international markets. Application of some compounds such as MeJA and ascorbic acid may affect growth characters and increase the total anthocyanin content of branches. Ascorbic acid as a water-soluble antioxidant in plants is a key factor in the detoxification of reactive oxygen species (Noctor and Foyer 1998). Numerous studies have focused on increasing ascorbic acid content to improve the nutritive value of plants and their susceptibility to stress (Gallie 2013). Higher levels of phytohormone eliminated radicals, which led to the maintenance of the plant’s defense function and improved growth (Smirnoff 2018). Furthermore, this compound stimulated cell division, promoted cellular growth and longevity (Horemans et al. 2000). Ascorbic acid is aslo used to improve vegetative growth and increase the quantity traits (such as soluble solids and anthocyanin) and quality of fruit such as peach (Shazly et al. 2013), grapevine (Fayed 2010), cupressus (Farahat et al. 2007) and apple (Alhevirin et al. 2016).

On the other hand, the jasmonic acid metabolites are critical plant phytohormones that regulate biotic and abiotic stress responses and mainly contribute to the growth processes of plants such as fruit ripening, pollen grain production and root growth (Popova et al. 1997; Babar et al. 2006; Nguyen et al. 2022). Furthermore, MeJA application can improve horticultural crop quality and enhance the total anthocyanin content (Rohwer and Erwin 2008). Although there is no research on the use of MeJA on red willow, several studies have demonstrated that plant growth and development were regulated in a coordinated method by the JA and IAA signal transduction pathways (Wang et al. 2020). The color of plants and fruits is determined primarily by anthocyanins, which belong to flavonoids. Following chlorophyll, these compounds serve as natural pigments in plants (Holton and Cornish 1995; Davies et al. 2012; Winkel 2001).

The existing literature on red willow failed to investigate the sole or combined effects of ascorbic acid or MeJA on this plant. Therefore, this study aimed to evaluate the effects of different concentrations of ascorbic acid and MeJA on the vegetative growth and some physiochemical traits of red willow.

Materials and methods

This study was conducted in Hossein Abad village, Markazi province, Iran (49° 30′27″E and 33°42′13″N) in 2019. The soil of the experimental area was clay-loam in texture with a pH of 7.9, containing total N (0.04%), total C (0.44%), and P (9.7 ppm), K (223.6 ppm), an EC of 1.6 ds cm−1. Red willow shrubs (3–4-year-old) were cultured in clay-loam soil and irrigated twice a week. The study was conducted as two separate experiments. The experimental treatments consist of MeJA foliar application at three levels (0, 100 and 200 mgL−1) and three concentrations of ascorbic acid (0, 100 and 200 mgL−1) by foliar spraying. In September and October, the juvenile branches were foliar sprayed twice in the morning, at two-week intervals. Several characteristics were evaluated, including the longest branch height, the nearest branch 1 and 2 height (i.e. the nearest two branches to the longest branch), total shrub diameter, diameter of the longest branch in three section (at lower, middle and upper parts), total anthocyanin content, salicin content, leaf chlorophyll (a, b and a + b) content, carotenoid, leaf length and width, leaves number on the longest branch and dry and fresh weight.

Photosynthetic pigments chlorophyll and total anthocyanin of branches were determined by Arnon (1967) and Wanger (1979) methods, respectively. The absorption rate of this material was measured at 550 nm using a spectrophotometer (the extinction coefficient of 330,000 Mm−1 cm−1). Salicin was also measured using a spectrophotometer (Measured according to TAPPI Code T204OM-88).

Branches were dried in the oven for 72 h at 60 °C to measure dry weight. Leaf length and width were measured with a ruler, and branch diameter with a caliper. The length and width of 10 leaves taken from the middle part of the longest branch in each treatment were measured. Furthermore, citric acid (0, 100 and 200 mgL−1) and ascorbic acid (0, 100 and 200 mgL−1) were used in the second experiment to evaluate phenotypic and biochemical characteristics.

Statistical analysis

This research was arranged in factorial based on a completely randomized design (CRD) with three replications. Data were analyzed by SAS software (Version 9.1). The means comparison was performed using Duncan's Multiple Range (DMRT) test at a probability level of 5%. Furthermore, a simple correlation was developed between total anthocyanin and morphological parameters using SPSS software (version 16).

Results and discussions

Analysis of variance results revealed that, the MeJA and ascorbic acid considerably affected the nearest branch 1, the height of the nearest branch 2, the leaves number in the longest branch, diameter (at lower, middle and upper parts) and the fresh and dry weights (P ≤ 0.01). Also, the height of the longest branch was affected by MeJA (P ≤ 0.01) and ascorbic acid (P ≤ 0.05) treatments. Furthermore, the interaction effect of ascorbic acid and MeJA was significant (P ≤ 0.01) on various parameters such as the nearest branch 1, height of the nearest branch 2, the number of the leaves in the longest branch, the diameter of the longest branch and fresh weight. Furthermore, significant differences were observed (P ≤ 0.05) for traits such as the longest branch height, dry weight, and total anthocyanin content. Ascorbic acid and MeJA, however, did not significantly affect the length and width of leaves on the longest branch, chlorophyll a, b, and a + b content, or carotenoid and salicin content (data not shown).

Shrub height

Results revealed that the MeJA foliar spray (200 mgL−1) combined with ascorbic acid increased the branches height (the longest branch, branches 1 and 2) (Tables 1 and 2). This result can be attributed to the MeJA contribution to increase the photosynthetic capacity, reduce sugars content, other carbohydrates, and organic acids in the treated fruit and plant (Janoudi and Flore 2003). Essentially, MeJA induces cellular division in growing tissues, which elongates cells and, consequently, lengthens stems (Creelman and Mullet 1997). This phytohormone is produced through lipoxygenases pathway and is known to regulate plant growth and environmental stress (González-Aguilar et al. 2006). Our findings were in agreement with the reports by Bidabadi et al (2013) who declared the positive effect of MeJA on the node numbers of grape cultivar. The current results are also consistent with those obtained for Rasheh and Red Bidaaneh cultivars, where MeJA (100 μM) enhanced leaves number (Sarabandi et al. 2019). On the other hand, some studies had exhibited that ascorbic acid affects the plasma membrane and protonic pumps. The acidic theory assumed that ascorbic acid stimulates weakening processes in the cell wall, causing it to expand and enlarge (Abdel-Aziz et al. 2009).

Table 1.

Means comparison of effects of methyl jasmonate foliar spraying on morphological and biochemical parameters of red willow shrub after one month

Methyl jasmonate
(mg L−1)		 Total anthocyanin
(mg/g fresh weight)		 Dry weight
(g)		 Fresh weight
(g)		 Total shrub diameter
(m)		 Middle diameter of the longest branch
(mm)		 Upper diameter of the longest branch
(mm)		 Lower diameter of the longest branch
(mm)		 Leaf number of the longest branch Height of the nearest branch 2
(cm)		 Height of nearest branch 1
(cm)		 The height of the longest branch
(cm)
0 0.000027b 8.13b 11.48b 1.36b 3.47c 1.16b 5.58b 31.66c 102.77c 129.55c 138b
100 0.000037b 8.42b 16.21a 1.49b 4.34b 1.42a 7.35a 41.55b 141.77b 156.55a 163.11a
200 0.000047a 16.41a 19.77a 2.09a 4.77a 1.52a 7.58a 50.44a 150.33a 144.44b 167.88a
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Similar letters in each column indicate no significant difference at 5% probability level

Table 2.

Means comparison of effect of ascorbic acid spraying on morphological and biochemical parameters of red willow shrub after one month

Ascorbic acid
(mg L−1)		 Total anthocyanin
(mg/g)		 Dry weight
(g)		 Fresh weight
(g)		 Total shrub diameter
(m)		 Middle diameter of longest branch
(mm)		 Upper diameter of the longest branch
(mm)		 Lower diameter of the longest
branch
(mm)		 Leaf number of the longest branch Height of nearest branch 2
(cm)		 Height of nearest branch 1
(cm)		 The longest branch height
(cm)
0 0.000028b 8.45b 9.81b 1.3c 3.76b 1.27b 5.7b 34.88b 111c 133.66b 146.22b
100 0.000033b 12.87a 15.2a 1.68b 4.55a 1.46a 7.25a 42.88a 135b 150.55a 160.33a
200 0.000046a 11.75a 16.31a 1.97a 4.28a 1.56a 7.55a 45.88a 148.88a 146.33a 162.44a
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Similar letters in each column indicate no significant difference at 5% probability level

Leaves number

The application of MeJA increased the leaves number on the longest branch (Table 1). The highest leaf number (50) was observed in the 200 mgL−1 concentration of MeJA treatment, while the lowest value (32) was recorded under the control conditions (0 mgL−1). In addition, ascorbic acid treatments increased the leaves' production compared with the control. This result highlights the key role of MeJA and ascorbic acid in regulating vegetative growth by increasing the number of leaves. Our findings are consistent with Salehi et al. (2016) who found that foliar spraying of ascorbic acids (100 mgL−1) enhanced the morphological and biochemical properties of petunia. As an antioxidant, ascorbic acid contributes significantly to the plant's growth. In other words, this compound is known as a growth-regulating factor with a dramatic influence on biological processes (Hendawy et al. 2010). MeJA also has growth-regulating properties and is involved in growth-related phenomena such as germination, seedling development, flower development, tuber production, twisting, leaf senescence, and fruit ripening. Furthermore, Jasmonate was found in high concentrations in the cell division regions of young leaves and reproductive structures (Wasternack and Hause 2002; Ananievaa et al. 2004). It has been reported that the use of MeJA in high concentrations activates the genes expression for photosynthesis, resulting in increased carbon fixation (Creelman and Muller 1997).

Diameter of the longest branch

Results specified that by increasing in concentrations of MeJA and ascorbic acid, the diameter of the longest branch (at upper, lower and middle parts) and the total diameter of the red willow shrub was enhanced. When compared with the control (0 mgL−1), this treatment at 200 mgL−1 concentration had the greatest effect on the mentioned traits (Tables 1 and 2). It can be assumed that MeJA and the ascorbic acid act as a growth stimulator, decreasing the environmental adverse conditions and stresses. Other studies have shown that MeJA increased growth through improving germination indices such as germination percentage and rate (Yusuf et al. 2012; Iqbal et al. 2012). Sarabandi et al. (2019) reported that MeJA treatment (200 μM) increased the stem diameter of two grape cultivars (Rashe and Red Bidaneh) which confirms the results of this study. Fresh and dry weights, leaves number, diameters, and heights were highest in the 200 mgL−1 concentration of MeJA treatment.

Fresh and dry weights

Similar to the previous results mentioned above, foliar spraying of MeJA and ascorbic acid positively affected red willow’s fresh and dry weight. Accordingly, these treatments at the 200 mgL−1 concentration led to the greatest fresh and dry weight (Tables 1 and 2). As mentioned before MeJA and ascorbic acid increased the height of the longest branch and leaves numbers. Therefore, increasing these vegetative organs can cause higher fresh and dry weight in the longest branch. These results are in line with a report on increasing stem diameter and leaves number by MeJA at a concentration of 100 μM on two cultivars of grape (Rashe and Red Bidaneh) (Sarabandi et al. 2019). Furthermore, Abdel-Aziz et al. (2009) declared that the application of ascorbic acid at concentrations of 50 and 100 mgL−1 improved the number of leaves, fresh and dry weights, and some chemical compounds of Syngonium podophyllum. Vitamins could enhance the fresh and dry weight of the plant organs which is not only due to higher water uptake (Nahed et al. 2007). Increased reductive sugars can considerably enhance photosynthetic pigment content, which will raise carbohydrate content (Abdel-Aziz et al. 2007). Moreover, ascorbic acid enhances the plant's ability to withstand chilling, drought, and salinity stresses (Hamada and AL-Hakimi 2009). Through interacting with cells and membrane lipids, ascorbic acid can play a decisive role in plant resistance against water loss and drought stress (Dolat-Abadian et al. 2010). Moreover, this vitamin has a significant effect on plant growth, cellular division, and nutrient cycle, as well as the electron chain system (Amin et al. 2008).

Total anthocyanin

Foliar spraying of ascorbic acid and MeJA enhanced the total anthocyanin contents compared with the control. Concentrations at 200 mgL−1 caused the highest enhancement in the total anthocyanin contents in the red willow branches bark (Tables 1 and 2). The positive effect of MeJA on growth traits appears to be due to its ability to further synthesize plant hormones, enzymes, as well as pigments such as anthocyanins (Capitani et al. 2005; Fariduddin et al. 2003). Studies have shown that the external application of MeJA can raise the accumulation of total anthocyanins in the plants (Kondo et al. 2001; Perez et al. 1997). The results of this research are in line with the reports on blackberry (Wang et al. 2008), strawberry (Ayala-Zavala et al. 2004), oriental lily (Taheri Shiva et al. 2013), apple (Rudell and Mattheis 2002) and raspberry (Ghasemnezhad and Javaherdashti 2008). Furthermore, these findings are in agreement with those obtained in peach fruit, which noted that 200 mgL−1 MeJA increased anthocyanin content (Pakkishet al. 2016). Additionally, Ghanati et al. (2010) found that although MeJA application at 100 μM increased marigold aerial anthocyanin content, this treatment had no significant effect at lower concentrations. In several plant species, MeJA treatment triggered anthocyanin accumulation, along with the expression of a gene series involved in the biosynthesis of anthocyanins (Sakamoto and Suzuki 2019). Our results revealed that the application of ascorbic acid also increased the total anthocyanin content of the shoots (Table 2). It has been identified that the ascorbic acid foliar spraying enhanced photosynthesis assimilates production and thus increased the amount of carbohydrates (Fayed 2010). Similarly, the positive effect of ascorbic acid on anthocyanin content can be explained by its ability to increase photosynthesis, stomatal conductance, and leaf surface (Zulaikha 2013). Additionally, ascorbic acid increases the activity of the PAL enzyme, which will increase the production of phenolic compounds along the phenylpropanoid pathway (Winkel-Shirley 2001). Farokhzad and Asghari (2016) identified that spraying Red Spar apple with 200 mgL−1 ascorbic acid improved the quality characteristics and coloration of this apple by increasing the total anthocyanin content. The same authors argued that the stimulation of apple fruit color can be attributed to PAL enzyme activity and its contribution to coloration by enhancing the synthesis of phenolic pigments such as anthocyanins (Farokhzad and Asghari 2016).

As for biochemical traits, MeJA increased the total anthocyanin. There is evidence that the application of MeJA, either individually or in combination, decreases chlorophylls and carotenoids such as in Helianthus annuus (Emery and Reid 1996), ‘Golden Delicious’ apple (Perez et al. 1993) and ‘Fuji’ apple (Rudell and Mattheis 2002). Accordingly, biochemical traits respond differently to MeJA. In other words, this study found that MeJA increased total anthocyanin content without affecting other biochemical traits.

Correlation among the morphological traits and total anthocyanin content

According to correlation analysis, the highest positive correlation was found between total anthocyanin content and leaf numbers on the longest branch (0.93), total shrub diameter (0.93), the height of nearest branch 2 (0.93), and fresh weight (0.89). The correlation between physiological traits and anthocyanin content (Table 3) supports the findings above. Moreover, there was a positive relationship between shrub diameter, leaf numbers, and finally fresh weight with total anthocyanin contents in the red willow branches. A shrub with a large diameter synthesized more anthocyanin due to its larger leaves and fresh weight (Asgari et al. 2021).

Table 3.

Correlation among total anthocyanin content and morphological characters in red willow

Anthocyanin The longest branch height Height of nearest branch 1 Height of nearest branch 2 Leaf number of the longest branch Lower diameter of the longest branch Upper diameter of the longest branch Middle diameter of longest branch Total shrub diameter Fresh weight Dry weight
Anthocyanin 1
The longest branch height − 0.04 ns 1
Height of nearest branch 1 − 0.05 ns 0.99** 1
Height of nearest branch 2 0.92** 0.039 ns 0.03 ns 1
Leaf number of the longest branch 0.93** 0.03 ns 0.03 ns 0.95** 1
Lower diameter of the longest 0.86* 0.04 ns 0.04 ns 0.98** 0.93** 1
Top diameter of the longest branch 0.52 ns 0.57 ns 0.57 ns 0.57 ns 0.59 ns 0.48 ns 1
Middle diameter of longest branch 0.76 ns 0.03 ns 0.04 ns 0.90* 0.94** 0.92** 0.55 ns 1
Total shrub diameter 0.93** 0.04 ns 0.04 ns 0.85* 0.93** 0.82* 0.45 ns 0.79 ns 1
Fresh weight 0.89* − 0.26 ns − 0.26 ns 0.91** 0.93** 0.91** 0.30 ns 0.89* 0.88* 1
Dry weight 0.73 ns 0.03 ns 0.037 ns 0.69 ns 0.86* 0.67 ns 0.42 ns 0.82* 0.89* 0.79 ns 1f
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Second experiment

Similar to the first experiment, results demonstrated that the ascorbic acid treatment (especially 200 mgL−1) significantly increased phenotypic traits such as the height of the highest branch, the height of the nearest branch 1 and branch 2, leaf number from the highest branch, total shrub diameter, diameter of the longest branch (at lower, middle and upper parts), fresh and dry weight of the longest branch and total anthocyanin content as a biochemical trait. However, the application of citric acid had no significant effect on evaluated morphological and biochemical traits (Table 4). According to these results, ascorbic acid considerably influenced morphological traits and the total anthocyanin content in red salix.

Table 4.

Means comparison of effect of ascorbic acid spraying on morphological and biochemical parameters of red willow shrub after one month in the second experiment

Ascorbic acid
(mg L−1)		 Total anthocyanin
(mg/g)		 Dry weight
(g)		 Fresh weight
(g)		 Total shrub diameter
(m)		 Middle diameter of longest branch
(mm)		 Upper diameter of the longest branch
(mm)		 Lower diameter of the longest branch
(mm)		 Leaf number of the longest branch Height of nearest branch 2
(cm)		 Height of nearest branch 1
(cm)		 The longest branch height
(cm)
0 0.000036b 5.55c 13.03c 1.30b 2.92b 1.11b 4.65c 92.22c 104b 110.55c 121.44b
100 0.000044b 8.90b 16.45b 1.44b 3.78a 1.56a 5.77b 111.44b 134.88a 131.88b 147.44a
200 0.000068a 15.17a 20.55a 2.07a 4.37a 1.50a 6.88a 142.66a 134.55a 140.55a 146.44a
Open in a new tab

Similar letters in each column indicate no significant difference at 5% probability level

Conclusions

Foliar application of 200 mgL−1 methyl jasmonate and ascorbic acid positively affected morphological traits and improved vegetative growth in red willow under field conditions. The progressive influence of methyl jasmonate on growth traits could be attributed to the synthesis of plant hormones, enzymes and the accumulation of pigments such as anthocyanins. By increasing total anthocyanin content, ascorbic acid contributes to better branch coloring. Additionally, this compound increases photosynthesis and carbohydrate production by increasing cell division, leaf number, and leaf fresh and dry weight in Hossein Abad village, Markazi province, Iran in Red willow. Additionally, a strong correlation was found between total anthocyanin and some phenotypic characteristics such as shrub height, diameter, leaf number, and fresh weight. Accordingly, it appears that selecting these characteristics could increase the crop yield.

Acknowledgements

This research was supported by the vice President Research and Technology of Arak University [Grant Number 97.15023, dated 2018/12/03], Arak, Iran, and is acknowledged by the author.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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