Effect of Exogenous Application of Several Plant Growth Regulators on Photosynthetic Pigments of Fennel Plants

In order to investigate the effects of some plant growth regulators on photosynthetic pigments and growth of fennel plants, a greenhouse experiment was conducted based on the randomized complete block design with three replicates in 2017. Treatments were the application of methyl jasmonate (25, 50, 100 and 200 μM), putrescine (0.25, 0.5, 1 and 2 mM) and 24Epibrassinolide at 0.001, 0.01, 0.1 and 1 μM and distilled water as a control. The results indicated that application of 0.5 Mm putrescine, exhibited significant effects on the chlorophyll a (62%), b (104%), total chlorophyll (72%), carotenoids (51%), flavonoids (51%), anthocyanin content (-14%), phenolic compounds (13%) and maximum quantum efficiency (17%) in dark condition and in light condition. Application of 24-Epibrassinolide resulted in a significant increase of chlorophyll a and total chlorophyll, carotenoids, phenol content, maximum quantum efficiency in the dark condition and photochemical quenching of fluorescence. The highest chlorophyll content and carotenoids were observed in treated plants with 0.1 μM 24Epibrassinolide, while the maximum phenol content was obtained by application of 0.01 μM 24-Epibrassinolide. The application of methyl jasmonate significantly affected the major chlorophyll and accessory pigments (except phenol) of fennel. Plants treated with 50 μM methyl jasmonate exhibited higher concentrations of chlorophyll a (3.25 mg per g FW), total chlorophyll (4.35 mg per g FW), carotenoids (0.87 mg per g FW) and flavonoids (4.75 μg per g FW). A significant dry weight increased after the application of methyl jasmonate and it can be concluded that the most effective treatment in this regard for fennel plants was 50 μM methyl jasmonate.


Introduction
Foeniculum vulgare Mill or fennel is an important medicinal plant belongs to the Apiaceae family.Fennel is originated from the Mediterranean area and widely been cultivated around the world (Mahfouz and Sharaf-Eldin, 2007;Aprotosoaie et al., 2010).Fruits and essential oil of fennel are used as flavouring agents in food, pharmaceutical products and cosmetic industry (Diao et al., 2014).Essential oil constituent of fennel contained mainly: estragole, transanethole, cis-anethole, fenchone and limonene (Díaz-Maroto et al., 2006;Rather et al., 2016).Fennel is recognized as well as an important plant in pharmaceutical and used as a stimulant, diuretic, sedative, digestive, carminative, antispasmodic and expectorant (Hashmi et al., 2012).
Polyamines (PAs) are small aliphatic amines, which behaved as a promoter of many plant physiological processes

Plant material and growth condition
A greenhouse experiment was conducted at the Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran, in 2017.
Greenhouse condition was related to a day temperature of 24 ± 2 °C, a night temperature of 16 ± 2 °C and a relative humidity of 50 ± 5%.
Each pot was filled with 20 kg sieved field soil.A soil sample taken prior to planting indicated soil pH (1:1, soil/H2O) was 7.9, cation exchange capacity (CEC) was 0.625 ds/m, and available P, K were 8.5 and 0.6 mg/kg, respectively.Ten seeds were planted in each pot and then thinned to five seedlings per pot once plants were wellestablished.
Treatments were arranged as a randomized complete block design with three replicates.Treatments application MJs include 25, 50, 100 and 200 μM, Put include 0.25, 0.5, 1 and 2 mM and BRs at 0.001, 0.01, 0.1 and 1 μM and distilled water as control were randomly assigned to the experimental pots.Treatments were imposed prior to the before flowering stage (65 days after planting) and 10 days after treatments imposed sampling for measured all parameters.

Determination of photosynthetic pigment
Chlorophyll a, b, total chlorophyll and carotenoids content were measured using the method described by Arnon (1967).For the extraction of pigments, 0.1 g of fresh tissue leaves were homogenized with acetone 80%, then the volume of the solution was reached to 20 ml by acetone 80%.Final solution centrifuged at 4,000 rpm at 10 min and optical absorption of the supernatant was performed in wavelengths 470, 645 and 663 nm.

Determination of anthocyanin and total phenol
For detriment anthocyanin content of fresh tissue, 0.1 g of leaves was homogenized with 10 ml ethanol acid (ethanol: HCl 99:1 v/v) and the solution was kept at in dark and 25 o C condition.Then solution centrifuged in 4,000 rpm at 10 min and absorbance was measured in wavelengths 550 nm.Anthocyanin content according to mg /g fresh weight (Wagner, 1979).
Total phenol content determined using Folin-Ciocalteu method described by Fletcher and Kott (1999).In brief, 0.1 g of leaves was homogenized with 10 ml ethanol 96%, then crude extract was allowed to stand for a further 24 hours in the dark.In finally, 1 ml ethanol 95%, 0.5 ml folin 50% and 1 ml sodium carbonate 5% addition to extract and the mixture was kept for 1 hour in dark condition.Absorbance was measured at 725 nm and total phenolic was calculated from the calibration curve, and the results were expressed as mg of gallic acid equivalent per g fresh weight.
such as cell division and tissue growth and development (Hasanuzzaman et al., 2014).Currently, putrescine, spermidine, and spermine are most well-known PAs.These compounds are organic polycations and exist in all organs of the plant, which have been associated with plant response to environmental stress (Alcázar et al., 2010).The PAs have substantial roles in DNA, RNA and protein synthesis (Igarashi and Kashiwagi, 2000).The PAs affect different plant developmental processes.For instance, Mahros et al. (2011), reported that the application of Put led to decline of pedicle and flower stalk length.However, Put significantly increased flower yield, chlorophyll a, b and the total carbohydrates.Likewise, photosynthetic pigments of African violet (Saintpaulia ionantha) were significantly increased by application of putrescine (Nanvakenary et al., 2013).In cucumber, exogenous application spermidine significantly increased chlorophyll contents and net photosynthetic rate (PN) and decreased constitutive loss processes (ФNO) and enhanced regulated nonphotochemical energy loss (ФNPQ) in the salt-stressed plants (Shu et al., 2012).
The MJs are found naturally in plants and they are responsible for regulation of many physiological and metabolic processes.Application of MJs improved plant resistance to various abiotic stresses (Bari and Jones, 2009).The MJs may influence Rubisco activity as well as the synthesis of other chloroplast proteins responsible for photosynthesis (Marín-Navarro et al., 2007).It was reported that MJs treatment of soybean appeared to arrest the growth, impaired leaf gas-exchange attributes and caused the loss of chlorophyll contents under water deficit conditions (Anjum et al., 2011).In another experiment, MJs increased leaf area, photosynthetic pigments and decreased chlorophyll fluorescence in Phaseolus coccineus plants (Hanaka et al., 2015).
The BRs have been recognized as polyhydroxysteroids, which play a fundamental role in many of the physiological and developmental processes of plants.It is reported that the BRs regulate seed germination, time of flowering, and maturation (Vardhini et al., 2006;Tang et al., 2016), Moreover, they are one of determining factor which confer plants resistance to environmental stresses (Fariduddin et al., 2014).Furthermore, BRs plays important roles in cellular responses such as stem elongation, pollen tube growth, leaf bending and epinasty, ethylene biosynthesis, xylem differentiation, and regulation of gene expression (Houimli et al., 2008;Hayat et al., 2012).Cevahir et al. (2008) reported that the application of exogenous BRs significantly affected the production of plant pigments such as chlorophyll, carotenoids, and anthocyanin in soybean.However, this effect was depended on the light condition.
Application of various plant growth regulators for modifications of plant physiological responses and alleviate abiotic stresses damages to plants has been well documented (Gururani et al., 2015;Mo et al., 2016;Awan et al., 2017).However, the time of PGRs application and eventual impact on photosynthesis behaviours of fennel plants is still elusive.This study was therefore conducted to study the photosynthesis potential and relative performance of fennel pigments obtained from PGRs treated plants.

Determination of flavonoids
The flavonoid was determined according to Krizek et al. (1998) methods, in order to 0.1 g from leaves extract in ethanol acid (ethanol: glacial acetic 99:1 v/v) and solution centrifuged in 3,600 rpm at 10 min.The supernatant was separate and allowed to stand for a further 10 min 80 o C. Finally, the absorbance was measured in three wavelengths 270, 300 and 330 nm.

Determination of chlorophyll fluorescence
Chlorophyll fluorescence was measured on the uppermost fully expanded leaf by using a fluorometer (model: OS-30P+ chlorophyll Fluorometer, Optic Science, USA).In this method, plants were adapted to darkness using dark adaptation clip for 15 minutes and then the fluorescence was measured in 1,000 µM photon m -2 s -1 by illumination of far-red light and calculated using the following equations (Arnon, 1949): In these equations, Fʹv/Fʹm is maximum quantum efficiency of PSII photochemistry in the light-adapted state, PSII equivalent maximum quantum efficiency of PSII photochemistry in the dark-adapted state, qP or photochemical quenching of fluorescence and NPQ is non-photochemical quenching.

Statistical analysis
Analysis of variance (ANOVA) was performed using Minitab, Ver, 16.The least significant difference (LSD) was used for mean separation and data is shown as a mean ± standard error (SE).Sigma plot v. 11 was used to calculate the regression equation.The reliability of models was evaluated using the coefficient of determination (R 2 ) and root-mean-square error (RMSE).In the present study, the parameters fitted by recon models (Table 1).

Effect of Put application on the fennel pigments and photosynthetic parameters
The results of the present study clearly showed that there was a significant effect of Put application on the chlorophyll a, b and total chlorophyll content of fennel leaves (Table 2).The Put application resulted in enhancement of chlorophyll content, which followed the Gaussian function.Leaves of treated plants with 0.5 mM Put showed the highest chlorophyll a, b and total content.Application of 0.5 mM Put increased chlorophyll a, b and total chlorophyll content by approximately 63%, 105%, and 73% respectively compared with control.Model analysis revealed that the maximum chlorophyll a, b and total chlorophyll were estimated by 0.65, 0.72 and 0.68 mM put respectively (Table 2).
Accessory pigments including carotenoids, anthocyanin, flavonoids and phenol content were significantly affected by Put treatments (Table 2).Results showed that the changes of anthocyanin and phenol due to the application of Put is followed a Gaussian function while other carotenoids and flavonoids were following log-normal function.The highest carotenoids content (0.82 mg per FW -1 ) and flavonoids (5.29 ug per FW -1 ) content was recorded at 0.5 mM Put, which approximately showed an enhancement about 50% and 51% over control.The maximum anthocyanin (11% higher than control) and phenol content (16% higher than control) was an observation of 0.25 and 1 mM Put respectively (Table 2).
The present results are in agreement with the results of previous studies which reported that there was a significant increase in total carbohydrates, chlorophyll a, b, carotenoids and PN in Chrysanthemum indicum and cucumber (Mahros et al., 2011;Shu et al., 2012).The Put might be involved in improving heat dissipation capacity and regulating the deepoxidation state of the xanthophyll cycle (Yuan et al., 2015).In addition, Put may improve the photosynthetic capacity of mesophyll cells by enhancing the carbon assimilation capacity (Araújo et al., 2011;Yuan et al., 2014).
Results of the present study showed that the application Put significantly affected the two parameters of chlorophyll fluorescence including PSII and F'v/F'm (Table 2).PSII and F'v/F'm in leaves of Put treated plants were higher than those of control plants.Maximum of PSII and F'v/F'm by change approximately 24% and 17% compared to control, were recorded when plants treated with 0.5 mM of Put.Log normal and Gaussian functions were provided the best fit to data obtained from PSII F'v/F'm traits of Put treated plants, respectively (Table 2).
Application of Put was not significant for fluorescence parameters of qP, and Fv/F0.Moreover, plant height and total dry weight were not affecting by the Put application (Table 2).Application of Put declined the ФNO and enhanced regulated ФNPQ in stressed plants (Nanvakenary et al., 2013).Some studies revealed that Put plays a role in protecting PSII against excessive energy through improving the thermal dissipation of the excitation energy (Rascher et al., 2000).The quantum functions of fennel leaves were significantly improved by Put application.The Put may be involved in alleviating the photoinhibition of UPSII and enhancing the photochemical quenching process (Yuan et al., 2014).
In addition, PAs can neutralize the negative charges of LHCII that, it is a putative site of qE, a rapid phase of NPQ, so that repulsion between different complexes is minimized (Navakoudis et al., 2007).

Effect of BRs application on the fennel pigments and photosynthetic parameters
Application of BRs exhibited a significant increase of chlorophyll a and total chlorophyll content compared to control while there was no significant effect on the chlorophyll b content (Table 3).Results showed that the highest chlorophyll content by approximately 133% and 154% of control treatment, obtained by the exogenous application of 0.1 µM BRs on fennel plants.The Lognormal function was capable to provide the best fit for the variations of chlorophyll content in treated plants.The slope of changes in chlorophyll a and total chlorophyll concentrations were estimated approximately 2.38 and 3.09 respectively (Table 3).
Among accessory pigments, only carotenoids and phenol content were influenced by the application of BRs.The accessory pigments of leaves were significantly improved by application of 0.1 and 0.01 µM BRs and the carotenoids and phenol contents increased about 43% and 11% of control treatments, respectively (Table 3).Furthermore, it was found that BRs improved chlorophyll fluorescence parameters in fennel leaves.Results showed that PSII and qP were significantly increased in BRs treated plants.The Log normal function provided the best fit for the PSII and qP parameters.Plants treated with 0.01 and 0.1 µM Put exhibited higher chlorophyll fluorescence 511 parameters such as PSII and qP, which were increased 25% and 11% higher than a control treatment (Table 3).
Although application of BRs did not significantly affects fennel growth, the overall trend of dry mater accumulation was followed a log-normal function.There are some reports showing that the application of BRs increases the chlorophyll, carotenoids and anthocyanin production in soybean (Cevahir et al., 2008).It has been reported that the exogenous application of BRs increased the pigment content in response to NaCl stress in some plants (Anuradha and Rao, 2003;Cevahir et al., 2008;Anuradha and RAO, 2011).Bajguz (2000) suggested that the BRs induced transcription and/or translation of the enzymes involved in chlorophyll biosynthesis linked with a decrease in the level of catabolizing enzymes.Piñol and Simón (2009) reported that the application of BRs led to palliative effects of terbutryn on fluorescence parameters, CO2 assimilation, and growth Vicia faba plants.It is suggested that the application of BRs could affect the terbutryn inhibition of PSII by displacement of QB from its binding site on the D1 protein of PSII.This protein is degraded when the photosynthetic system cannot process the energy of accumulated photons but little is known about the PSII repair process, either at the level of protein synthesis, insertion, and concomitant assembly of the D1 protein or at later functional post-translational assembly steps (Zhang et al., 2000).Thus, the D1 protein of PSII must be degraded, resynthesized de novo, and reinserted into the PSII reaction centre to repair the damage and re-establish PSII function (Piñol and Simón, 2009).It is known that BRs could influence the gene expression and protein synthesis.Thus, BRs could be implicated in the control of D1 damage and repair (Wang et al., 2006).

Effect of MJs application on the fennel pigments and photosynthetic parameters
It was cleared that the exogenous application of MJs significantly affected the major chlorophyll and accessory pigments (except phenol) of fennel (Table 4).Lognormal provided the best fit for changes in major chlorophyll, carotenoids and flavonoids, whereas the Gaussian function well applied to describe the changes of anthocyanin in the MJs treated fennel leaves.The result indicated that exogenous MJs affect the chlorophyll concentration in treated plants.The maximum and minimum slope of variations in photosynthetic pigments was obtained 0.78 and 0.53 for chlorophyll a and b, respectively (Table 4).Application of MJs enhanced total concentrations of the chlorophyll and accessory pigments.In comparison with control plants, treated plants with 50 µM MJs exhibited 91%, 95%, 59%, and 36% chlorophyll a, total chlorophyll, carotenoids, and flavonoids, respectively (Table 4).Marín-Navarro et al. (2007) reported that MJs are regulators of rubisco activity as well as the synthesis of other chloroplast proteins responsible in photosynthesis.
Also, MJs treatment appeared to arrest the growth, impaired leaf gas-exchange attributes and caused the loss of chlorophyll contents of soybean plants under water deficit (Anjum et al., 2011).It is reported that the MJs increased leaf area, photosynthetic pigments and decreased chlorophyll fluorescence in Phaseolus coccineus plants (Hanaka et al., 2015).Wierstra and Kloppstech (2000) reported that MJs declined the light-harvesting complex II (LHC II) and small subunit of Rubisco (SSU) transcript and D1 protein levels.The reduction in D1 levels of MJs treated leaf-segments might be due in part to the diminished carotenoids content as β-carotene is required for the assembly of D1 protein into functional PSII reaction centres.It is suggested that MJs in developing leaves might play a role to prevent the formation of premature photosynthetic structures and accumulation of nutritive storage proteins for further leaf development.
Based on the obtained results, it is noted that among all of chlorophyll fluorescence parameters, only PSII, and qP were significantly affected by MJs (Table 4).The MJs increased these parameters, while the highest amount PSII, and qP by was observed in 50 µM MJs (increased by 24% and 8% compared to control).The log-normal function was appropriate to describe the variation of PSII, and qP in treated plants with MJs (Table 4).Jin et al. (2011) showed that the foliar spraying of 0.2 and 0.5 mmol MJs exhibited the positive effects on mitigating the decrease of Fv/Fm, PSII, and qP and the increase of qN under drought stress.It is suggested that foliar application of MJs could alleviate the degradation of chlorophyll and play a definite role in protecting the PSII under drought stress, decrease the damage of drought stress on the seedlings, promote the rapid MJs recovery of chlorophyll fluorescence parameters after re-watering, and thus, ensure the regrowth of fluecured tobacco seedlings.Wierstra and Kloppstech (2000) stated that the increase of photochemical efficiency of PSII by MJs leads to an equilibrium between D1 destruction and repair which depends on the light intensity and a restoration phase during which PSII activity is recovered due to the integration of the newly formed D1 protein.
Application of MJs significantly increased the total dry weight of fennel plants, while this was not significant for plant height (Table 4).Change in plant dry weight was well explained by Gaussian function, with the slop of 17.8 and in 61 µM MJs reached to maximum dry content.

Conclusions
The study proved that the effects of PGRs on major chlorophyll was more pronounced compared to accessory pigments, although among all the chlorophyll fluorescence parameters, only PSII was influenced by all of the PGRs.The present findings showed that the exogenous application of 0.5 mM Put, 0.1 μM BRs and 50 μM MJs provided the optimum concentrations of PGRs to improve photosynthetic indices of fennel plants.Among PGRs, only MJs exhibited the significant effects on the growth and development of fennel.

Table 1 .
Models used to fit parameters measured of fennel

Table 2 .
Effect application of Put concentration on photosynthetic pigments, chlorophyll fluorescence parameters and biomass of fennel (Foeniculum vulgar Mill) leaves

Table 3 .
Effect application of BRs concentration on photosynthetic pigments, chlorophyll fluorescence parameters and biomass of fennel (Foeniculum vulgar Mill) leaves