6-Benzylamino purine outperforms Kinetin and Thidiazuron in ameliorating flower longevity in Calendula officinalis L. by orchestrating physiological and biochemical responses

In view of extending the relatively brief postharvest life of flowers by a range of technologies, the present study elucidates the implication of 6-benzylamino purine (BAP), kinetin (KN) and thidiazuron (TDZ) on postharvest performance and flower longevity of isolated flowers of Calendula officinalis. BAP and KN belong to adenine group cytokinins while as TDZ is a diphenyl urea compound having cytokinin like activity. The harvested flowers were supplemented with BAP, KN and TDZ at various concentrations viz., 25, 50, 75 and 100 μM at one day before anthesis (cup shaped) stage. The control was designated by a distinct set of flowers held in distilled water (DW). Our findings revealed substantial enhancement in flower longevity by application of various growth regulators as compared to the control. Vase solutions containing BAP and KN at 50 μM and TDZ at 75 μM (individually) were most effective in improving the longevity of cut Calendula flowers. Improvement in flower longevity was primarily associated with high membrane stability index (MSI), upregulated activities of various antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX), besides an attenuated lipoxygenase (LOX) activity in the petals. As compared to control, the treated flowers exhibited higher values of soluble proteins, total phenols and total sugars, besides lower α-amino acid content in the petal tissues. However, BAP outplayed TDZ and KN in improving the flower longevity of Calendula officinalis by maintaining higher physiological and biochemical stability in petals.


Introduction
Flowers offer an exclusive window to unveil the physiological, biochemical and molecular changes that occur in cells and tissues while progressing towards senescence. Senescence is an intricate process encompassing systematic and highly co-ordinated phases of inception, progression and completion implicated in an extensive re-programming of gene expression. The process involves shut down of several biosynthetic pathways and up-regulation of many degradative pathways, eventually leading to the cell death (Saeed et al., 2014;Ahmad and Tahir, 2016;Sarwat and Tuteja 2019). In addition to alterations in molecular crosstalk, the flower senescence involve various physiological and biochemical changes such as loss of water from the senescing tissue, leakage of ions, generation of reactive oxygen species (ROS), increase in membrane fluidity/lipid peroxidation, hydrolysis of proteins, nucleic acids and carbohydrates (Shibuya et al., 2016;Salleh et al., 2016;Chen et al., 2018;Hemati et al., 2019). Visible symptoms of petal senescence typically include colour change, rolling and wilting (Bagniewska-Zadworna et al., 2014), while anatomical changes such as collapse of mesophyll cells occurs well before the apparent morphological changes (Wagstaff et al., 2003;Shibuya et al., 2016).
Flower longevity and quality are important factors that determine the economic and market value of ornamental flowers (Hegazi, 2016). The key to extend the flower longevity is to understand the biological processes governing postharvest physiology, respiration, transpiration, hormonal imbalance and the activation of enzymes associated with petal discoloration (Wojciechowska et al., 2018). Cytokinins act as the front line anti-senescence hormones that improve the longevity in both the ethylene sensitive and insensitive cut flowers (van Doorn and Woltering, 2008;Ahmad and Tahir, 2016;Iqbal et al., 2017). The anti-senescence role of cytokinin make it the hormone of considerable demand in the postharvest technology to improve the vase performance of cut flowers. Cytokinin delay senescence by increasing sink activity, promote transport and maintain increased amounts of metabolites in flower petals, besides preventing lipid peroxidation of membranes (Radio et al., 2017;Iqbal et al., 2017). Cytokinins may alternatively extend the longevity of flowers by enhancing the levels of antioxidant compounds such as phenols and anthocyanins, which function as scavengers of ROS produced during petal senescence (Trivellini et al., 2015). Molecular studies report that cytokinins defer senescence through autoregulation of cytokinin biosynthesis through isopentenyl transferase gene using a promoter senescence-associated gene 12 (SAG12). In this regard, SAG12:IPT gene has been extensively evaluated in different species, and all of them evidenced delayed senescence (Xiao et al., 2017).
Calendula officinalis (marigold) of asteraceae (compositae) family is grown worldwide as an ornamental plant with great potential in floriculture industry as cut flower because of its vivid and attractive colours (Chopde et al., 2015). However the brief postharvest life of the flower diminishes its potential market value. Calendula is least sensitive to ethylene (Kondo et al., 2017). Pertinently, oxidative stress is reported as the main cause of limited vase life in ethylene insensitive flowers (Rahmani et al., 2015) and implication of cytokinins on oxidative stress underlying flower senescence in Calendula is yet to be explored. In this regard, an experiment was set-up to ascertain the implication of adenine type cytokinins viz., benzylamino purine (BAP) and kinetin (KN) and a diphenyl urea compound; thidiazuron (TDZ) having cytokinin like activity on oxidative stress associated with flower senescence in C. officinalis by evaluating various physiological and biochemical parameters with the ultimate aim to improve its postharvest performance and flower longevity.

Plant material and treatments
Fresh flower buds of Calendula officinalis were harvested at 900 h from Kashmir University Botanical Garden (KUBG) at one day before anthesis stage. After transported to the laboratory, the flowers were re-cut to a uniform stalk length of 4 cm under distilled water and divided into four sets. Three sets were supplied with BAP, KN and TDZ (individually) of different concentrations viz., 25, 50, 75 and 100 µM. Another set of flower buds held in distilled water (DW) designated the control (Figure 1). Each treatment was represented by ten replicates. The experiment was conducted under controlled conditions with relative humidity (RH) of 60%-70%, 12 h light period a day and average temperature of 21 ± 2 °C . The first day of experiment when flowers were subjected to different treatments was designated as day zero. Visual assessment and changes in various parameters occurred in the flowers during the experiment were recorded on day 2 and 4 of transfer of flower buds to the respective vase solutions.

Evaluation of flower longevity and floral diameter
The average flower longevity of the cut flowers was calculated from second day of experiment until the last flower lost its market value. The floral diameter was measured as average of two perpendicular measurements across the floral head on day 2 and 4 of the experiment.

Evaluation of membrane stability index (MSI)
The MSI of petal tissue evaluated in the form of electrolyte leakage was determined by employing Sairam (1994) method. 0.5 g of petal tissue was incubated in 25 ml of ion free water at 25 °C for 30 minutes and 95 °C for 15 minutes. The conductivities of the petal tissue incubated at 25 °C (C1) and 95 °C (C2) were obtained on Elico CM180 conductivity meter. MSI was calculated by using the formulae: Determination of total phenols and sugar fractions 1 gram chopped petal tissue from each treatment was macerated and centrifuged. An appropriate aliquot taken from the supernatant was used to estimate these parameters. Swain and Hillis (1959) method was used for quantification of phenols using gallic acid as standard. Nelson's (1944) protocol was employed for quantification of reducing sugars using glucose as standard. Estimation of total sugars was determined by converting non-reducing sugars into reducing sugars using invertase enzyme. Amount of non-reducing sugars was determined by evaluating the difference between total and reducing sugars.

Quantification of soluble proteins and α-amino acids
For quantification of proteins, 1 gram of petal tissue was macerated in 100 mM phosphate buffer (pH 7.2) comprising NaCl (150 mM), ethylene diamine tetraacetic acid (1 mM), Triton X-100 (1%), glycerol (10%), polyvinyl pyrrolidone (10%) and Dithiothreitol (1 mM). The mixture was subjected to centrifugation at 12,000 g at 4 °C in a refrigerated centrifuge for 15 minutes. Employing Lowry et al. (1951) protocol, a suitable aliquot taken from the supernatant was used for quantifications of proteins. Estimation of α-amino acids was performed by employing Rosen's (1957) protocol using glycine as standard.
Extraction and determination of enzyme activities Superoxide dismutase (SOD) SOD activity was determined by observing the inhibition of photochemical reduction of nitroblue tetrazolium (NBT), employing Dhindsa et al. (1981) protocol. The reaction mixture contained sodium carbonate (50 mM), NBT (75 µM), ethylene diamine tetraacetic acid (0.1 M), methionine (13 mM) in 50 mM phosphate buffer (pH 7.8) and 0.1 ml of the enzyme extract in a final volume of 3 ml. The reaction was initiated with the addition of 2 µM riboflavin and keeping the test tubes in water bath at 25 °C and illuminating with a 30 W fluorescent lamp. The test tubes were kept in dark after the reaction was stopped by switching off the light. Other identical unilluminated test tubes served as blanks. Absorbance was obtained at 560 nm by using spectrophotometer. One unit of SOD activity was defined as the amount of the enzyme that prevents 50% photoreduction of NBT to blue formazan in comparison to the reaction mixture kept in dark. The SOD activity was expressed as units min -1 g -1 protein.

Catalase (CAT)
CAT activity was assayed by Aebi (1984) protocol. Petal tissue (1 g) was macerated and homogenized in 100 mM potassium phosphate buffer of neutral pH containing ethylene diamine tetra acetic acid (1 mM). The reaction mixture contained 50 mM potassium phosphate buffer of neutral pH, H 2 O 2 (12.5 mM), enzyme extract (50 µl) and distilled water to make the volume to 3 ml. With the addition of H 2 O 2, reaction was initiated and the catalase activity was assayed by determining H 2 O 2 consumption for 3 minutes at 240 nm and expressed as µM H 2 O 2 red. min -1 mg -1 protein. Chen and Asada (1989) protocol was employed for determining APX activity. Petal tissue (1 gram) was macerated in 100 mM sodium phosphate buffer containing ascorbate (5 mM), glycerol (10%) and ethylene diamine tetraacetic acid (1 mM). The APX activity was determined in 1 ml reaction mixture containing 50 mM potassium phosphate buffer of neutral pH, ascorbate (0.1 mM), H 2 O 2 (0.3 mM) from absorbance decrease due to ascorbate oxidation. The absorbance was recorded at 290 nm for 3 minutes and expressed as µmmin -1 mg -1 protein.

Statistical Analysis
A completely randomized design was performed on the data collected from experiment. The investigation was repeated twice to minimize the variance between the variables. Complete statistical analysis was done and Least significant difference (LSD) was computed at P 0.05 using SPSS ® (SPSS version 16 software; Chicago USA).

Results
Based on the present investigation, significant differences were revealed among the growth regulators (KN, BAP and TDZ) tested at various concentrations in extending the longevity of cut Calendula flowers. The prolonged flower longevity commensurate with up-regulated activities of antioxidant enzymes, high membrane stability, elevated total phenols, total sugars and soluble proteins, besides a decreased α-amino acid content and LOX activity in the tissue samples. Application of BAP and KN at 50 µM and TDZ at 75 µM resulted in maximum enhancement in flower longevity and improved postharvest performance of cut Calendula flowers considerably. The petal tissue from the samples treated with the concentrations above and below of optimal concentrations (50 µM BAP and KN) and (75µM TDZ) showed a significant decrease in all the parameters investigated except LOX activity and α-amino acids which showed a marked increase. However, BAP profoundly increased the flower longevity and was most effective in improving postharvest performance of Calendula flowers as compared to other growth regulators and control.

Flower longevity and floral diameter
Flower senescence was characterized by loss of petal turgidity leading to shrinkage and upward erection of petals in the form of needles. The petals however didn't abscise, remained on the flower pedicels till complete wilting. The flower buds held in distilled water (control) lasted for 5 days only, whereas application of growth regulators increased the longevity of cut Calendula flowers markedly. The longevity of flowers held in 50 µM BAP, 75 µM TDZ and 50 µM KN solutions recorded was 13,10 and 8 days respectively, revealing BAP as most efficacious cytokinin in improving the longevity of cut Calendula flowers ( Figure 2).
Head diameter of flowers held in various test solutions showed a significant increase as compared to control which however was observed to decrease gradually from day 2 to 4 ( Figures 3a, b).

Membrane stability index (MSI)
Changes in MSI values suggest that with the development of flowers, membranes lose their integrity resulting in outpouring and loss of intracellular compartmentalization. Selected growth regulators in this study revealed their effectiveness in protecting cell membranes. Highest MSI value was found in the flowers treated with BAP, while those treated with TDZ and KN registered marginally lower values but significantly higher than the control. However, membrane stability in petal tissues was found to decrease gradually from day 2 to 4 as evident from MSI values in Figure 4.

Total phenols and sugar fractions
As compared to control, higher phenolic and sugar (total, reducing and non-reducing sugar) content was observed in the flowers held in BAP followed by TDZ and KN (Figures 5a, b). Furthermore, reducing sugar content was found marginally higher than non-reducing sugar content in all tissue samples treated with various growth regulators (Figures 5c, d). However, a significant reduction in both of these parameters was recorded in the flower petals with progression in time from first to second analysis.

Soluble proteins and α-amino acid content
Flowers treated with various growth regulators registered higher content of soluble proteins in petal tissues. The petal tissue from the samples treated with BAP showed maximum enrichment of soluble proteins, besides higher reduction in the α-amino acids followed by TDZ and KN. Maximum α-amino acid content was recorded in the samples of the control. However, soluble protein content was found to decrease with progression in time from day 2 to 4 with the concomitant increase in α-amino acids (Figures 6a, b).

Activities of antioxidant and lipoxygenase enzymes
SOD and CAT activities were profoundly augmented in the flowers treated with BAP followed by TDZ and KN, whereas TDZ was more effective in enhancing the APX activity in the tissue samples (Figures 7a, b, c).   Furthermore BAP application resulted in considerable decrease in LOX activity followed by TDZ and KN (Figure 7d). Least antioxidant enzyme activities (SOD, CAT and APX) were assayed in untreated petal tissues (control), besides an increased LOX activity. Activities of these enzymes were found to decline as the flower development progressed with concomitant increase in LOX activity. However, 50 µM BAP was found highly effective in preventing the down-regulation of activities of these enzymes in the petal tissues as pictured in Figures  7a, b, c, d.

Discussion
Flower longevity is one of the most important characteristics of cut flowers, which is shortened by a number of factors varying in wide range of ornamentals (Vehniwal and Abbey 2019). The current study ascertains the role of BAP, TDZ and KN (as continuous treatments) in regulating senescence and mitigating postharvest losses in cut flowers of Calendula officinalis, keeping in view its immense demand in floriculture industry as cut flower. Although, TDZ doesn't belong to BAP and KN type of cytokinins (adenine type), but it possesses cytokinin like activities, so the three tested growth regulators are discussed together as cytokinins.
Exogenous application of cytokinins and TDZ increased the longevity of cut Calendula flowers significantly. Cytokinins could improve flower longevity by affecting many physiological processes such as free radical formation, membrane stability, water balance and protein metabolism ( Each value denotes the mean of three replicates for each treatment. establishes a water potential gradient, leading to entry of water into petal tissues making them turgid, consequently increasing the floral head diameter (Reid and Wu, 2018). Calendula is least sensitive to ethylene, and oxidative stress is known to be the main reason of early senescence in ethylene insensitive flowers. Against to this, BAP was highly effective in maintaining a decreased LOX activity in flowers followed by TDZ and KN, which prevented the excessive lipid peroxidation of the membranes as evident from increased MSI values in the tissue samples. The reduced LOX activity resulted in maintenance of adequate phospholipids, proteins and thiols by preventing the seepage of proteases from vacuoles into the cytoplasm (Macnish et al., 2010;Liu et al., 2016a;Dek et al., 2017). Cytokinin mediated enhanced membrane integrity can also be attributed to its involvement in maintenance of high water content, antioxidant system and reduced peroxidation rates in tissues, which are greatly affected by the stresses like senescence Honig et al., 2018). Studies on senescence regulation in C. officinalis, showed that BAP could minimize the reduction in MSI values (Kaur et al., 2015). Moreover, application of BAP to cauliflower and carnations resulted in a marked decrease in lipid peroxidation with concomitant increase in membrane stability of tissue samples (Siddiqui et al., 2015;Ramtin et al., 2019).
High protein levels with concomitant lower α-amino acid content was maintained in flowers supplied with various growth regulators particularly in BAP treated flowers. Application of postharvest preservatives that downregulate the protease activity can be suggested as a strategy for extending the vase life and minimizing postharvest losses of flowers (Dwivedi et al., 2016). External application of cytokinins was found very effective in delaying senescence of various cut flowers such as Iris, Phalaenopsis and Nicotiana by arresting degradation of proteins and chlorophyll Chen et al., 2018;Tahir et al., 2019). Indeed, cytokinins extended the longevity of Andrepogon and Nicotiana flowers not only by preventing protein degradation but also by inhibiting the activity of proteases. This suggests that cytokinins delay senescence by downregulating the programmed cell death due to prevention of upsurge of proteasomes (Macnish et al., 2010;Nisar et al., 2015). Exogenous application of BA strongly upregulated some heat shock proteins (HSPs) in petunia flowers which might protect cellular functions or slow down the action of proteases (Veerasamy et al., 2007).
Cytokinins activate defense mechanism against postharvest senescence additionally through enrichment of phenols (Schmitzer et al., 2010;Ahmad and Tahir, 2017) as ascertained during the present study. Phenol enrichment has been reported to counter endogenic perturbations, biotic and abiotic oxidative stress in various flowers (Lattanzio et al., 2006;Ahmad and Tahir, 2017). Cytokinin mediated accumulation of phenolic compounds may contribute in strengthening cell wall structures and prevent oxidative damage to membrane lipids and proteins by modulating the peroxidation kinetics through fee radical scavenging (Soleimani Aghdam et al. 2015). A higher phenolic content was assayed in the petal tissue from the samples treated with various growth regulators. Petunia flowers treated with cytokinins also showed a considerable enhancement in phenol accumulation (Trivellini et al., 2015). In Arabidopsis, exogenous application of cytokinins resulted in increased phenol accumulation by stimulating the activation of phenylalanine ammonia lyase (PAL1), chalcone synthase and chalcone isomerase genes, involved in the synthesis of various phenolic compounds (Diekman and Hammer, 1995).
Higher sugar content was maintained in the tissue samples treated with various cytokinins consistent with the earlier findings which reported a correlation between carbohydrate accumulation and flower longevity by cytokinin application in various flowers such as Gladiolus, Iris, Nicotiana, Gerbera, and Rosa (van Doorn and Woltering, 2008;Ahmad et al., 2018;Tahir et al., 2019;Hemati et al., 2019;Ramtin et al., 2019). Cytokinins maintain high sugar content in petal tissues by enhancing the sink strength, thus inhibiting the mobilization of sugars from the floral parts to ovary during nutrient remobilization (van Doorn and Woltering, 2008). Furthermore, flower petals like other terminally differentiated sink tissues often contain highly active invertase enzyme, that catalyzes the hydrolysis of sucrose (non reducing sugar) into glucose and fructose (reducing sugars) (Woodson and Wang, 1987).
An increase in ratio of non-reducing to reducing sugars occurs concomitant with petal senescence (Nichols, 1973). Against to this, cytokinins maintained marginally high ratio of reducing to non-reducing sugars in the petal tissues of Calendula flowers, contributing to its prolonged vase life.
As mentioned above, oxidative stress caused by production and accumulation of ROS has been recognised as main factor for early postharvest senescence in ethylene insensitive flowers (Rahmani et al., 2015;Dwivedi et al., 2016;Saeed et al., 2016). In order to combat adverse effects of oxidative stress, high antioxidant enzyme activity is prerequisite for efficient scavenging of excessive ROS, that otherwise damage cell membranes and cause flower deterioration. An upregulated activities of SOD, CAT and APX enzymes were recorded in petal tissues supplemented with BAP, KN and TDZ. The association of high antioxidant enzyme activity and flower longevity has been reported in several cut flowers such as carnations, chrysanthemums, red dragon, gerbera, gladiolus and iris (Dwivedi et al., 2016;Ahmad and Tahir, 2016;Naing et al., 2017;Hemati et al., 2019).
Cytokinins improve flower longevity either directly by scavenging the free radicals or alternatively by upregulating the production and activities of antioxidant enzymes that scavenge ROS (Siddiqui et al., 2015;Liu et al., 2016b;Danilova et al., 2017). Petals with decreased APX activity has been found to show early senescence symptoms in various flowers like daylily, gladiolus and carnation by generation and accumulation of ROS (Mittler et al., 2004;Saeed et al., 2014). Exogenous application of cytokinins effectively reduced the free radical formation in tobacco plant during senescence (Dek et al., 2017). The role of cytokinins in maintaining the ambient levels of antioxidant enzymes for longer vase life of the flowers has also been demonstrated by application of cytokinin oxidase inhibitors in wall flowers (Bartrina et al., 2017).

Conclusion and future scope
From the present investigation, the authors conclude that treatment of isolated flower buds with adenine type cytokinins (BAP and KN) and a diphenyl urea compound (TDZ) having cytokinin like activity minimized the postharvest oxidative damage in C. officinalis as evident from improved flower longevity and postharvest attributes. BAP was found most efficacious and outcompeted other growth regulators in improving the postharvest performance and longevity of flowers. Based on the findings, it can be argued that the improvement in longevity and quality of isolated flowers of C. officinalis by application of BAP, TDZ and KIN could be attributed to up-regulated activities of various antioxidant enzymes, upsurge in MSI and phenolic content, besides lower LOX activity and α-amino acid content in the petal tissues. Furthermore, maintenance of elevated content of soluble proteins and sugar fractions in the petals by these growth regulators also qualify their role in improving the flower longevity of Calendula officinalis.
The study offers immense future scope to mitigate the flower senescence and postharvest losses at molecular level, which will argument the studies of complex interplay and crosstalk of cytokinins and related compounds in this beautiful and fascinating flower. Moreover, studies at molecular level may prove beneficial in gaining an insights into the strategies of flower senescence across the family. Understanding the mechanism of flower senescence in this flower will unveil strategies to modulate it by various chemical formulations and/or molecular interventions for its efficient and productive postharvest output.