Introduction

Senescence, a genetically programmed event represents the final phase in ontogeny of flowers in which a sequence of irreversible events are initiated, inevitably leading to cellular breakdown and organ death. The process is orchestrated by changes in the levels of growth regulators and their crosstalk acts as regulatory signals for the commencement or cessation of specific reactions. The biochemical and physiological indices that affect the postharvest flower senescence include; water uptake, lipid peroxidation rates, membrane stability, respiratory activity, protein and sugar content, besides activities of antioxidant enzymes in the petal tissues (Reid and Jiang 2012; Rani and Singh 2014; Hemati et al. 2019). Neck (stem) bending has been reported as a primary reason for limited life of many cut flowers which precedes well before the wilting of the petals (Naing et al. 2017; Gómez-Merino et al. 2020). It is caused by insufficient mechanical support in the xylem, inadequate lignin rich sclerenchyma cells in the flower stems and microbial contamination (van Doorn and de Witte 1994; Williamson et al. 2002; Perik et al. 2012; Naing et al. 2017; Shabanian et al. 2018).

From villain to hero to biochemistry’s new superstar, NO has emerged as a novel molecule regulating diverse set of biological processes in both animal and plant kingdom (Moroz et al. 2020; Verma et al. 2020). This enigmatic, though unique transportable multidimensional signaling molecule due to its lipophilic nature plays pivotal role in regulating various processes in plants such as resistance to untoward environmental stresses, defense responses, hormonal modulation and programmed cell death (Shi et al. 2016; Ahmad et al. 2018; Kumar Rai et al. 2018). NO is involved in the broad spectrum of physiological and developmental processes and in multiple modes of action associated with flower senescence (Lamattina et al. 2003; Hasanuzzaman et al. 2016). It has been well-documented that NO functions as a negative regulator during flower senescence hence acts as a novel substitute for some other toxic chemicals like silver thiosulphate (STS) in postharvest technology (Procházková and Wilhelmová 2011; Rabiei et al. 2019; Deng et al. 2019). Exogenously applied NO has significantly increased the vase life of various cut flowers such as gerbera, iris, tulip, snapdragon, delphinium, oriental lily, rose, gladiolus, chrysanthemum and carnations (Badiyan et al. 2004; Naing et al. 2017; Kazemzadeh-Beneh et al. 2018; Zhang et al. 2018; Deng et al. 2019). Indeed application of NO has been shown to extend not only the longevity of cut flowers but also the shelf life of various fruits, e.g. peach, cherry (Saba and Moradi 2017; Rabiei et al. 2019) and vegetables, e.g. broccoli (Shi et al. 2016). NO has been found to delay senescence at lower optimal concentration, while its higher concentration (super-optimal) reportedly causes nitrosative damage to plants and provokes early senescence (Naing et al. 2017; Sami et al. 2018).

Calendula (Marigold) of Asteraceae family is one of the leading commercial cut flowers in the world securing top ranks in global floricultural industry. Being prolific and floriferous, besides having medicinal and culinary importance, their pungent blooms bring bursts of sunshine into the room and smiles to the face. However, short flower longevity due to early neck (stem) bending and associated physiological and biochemical instability reduces its potential export and trade value. Despite the application of various anti-senescence agents to combat such problems, the role of NO remains unexplored till date for this flower. Calendula is insensitive to ethylene (Kondo et al. 2017). Pertinently, NO is highly efficacious in flowers  in which senescence progresses independently of ethylene (Woltering and Van Doorn 1988; Dwivedi et al. 2016). It is in this perspective that the study was undertaken to ascertain the implication of SNP as a source of NO on neck bending associated with senescence and postharvest performance in flowers of C. officinalis by investigating various physiological and biochemical parameters with the particular aim to ameliorate the longevity of  these joyful and bright flowers.

Materials and methods

Plant material and SNP treatment

Fresh uniform isolated flower buds of C. officinalis grown in Kashmir University Botanic Garden (KUBG) were utilized for this study. The flowers at one day before anthesis stage were harvested at 9:00 h and quickly transported to laboratory in distilled water. The flower stems were re-cut to an approximate 8 cm length in accordance with marketable size. Two flowers of approximately same size were placed in 100 ml Ehrylenmeyer flasks (vases) filled with distilled water containing different concentrations of SNP viz., 50, 100, 150 and 200 µM. Each treatment had ten replicates (flasks) wrapped with aluminum foil in order to avoid photo-degradation of SNP (release of a nitrosyl ligand and cyanide ion). A distinct set of flowers held in distilled water served as control. The mouth of each flask was sealed with parafilm to keep the flowers upright and to avoid evaporation. The day of application of SNP treatments to isolated flowers was designated as day zero. The laboratory conditions were adjusted with relative humidity of 55 ± 10% and 12 h light period a day and mean temperature of 22 ± 2 OC. Visual assessment of flowers was performed from day zero till last day of experiment and changes in various parameters were recorded on day 2 and 5 of application of different treatments.

Measurements

Flower longevity and floral diameter

The longevity of cut Calendula flowers was calculated from the second day of experiment (day 1) when almost all the flowers of the treatments were opened and assessed to be expired when last flower displayed signs of senescence and lost its decorative/market value. The floral diameter was evaluated as the mean of two perpendicular measurements of flower heads.

Bacterial density, solution uptake and neck bending

The bacterial density was determined by recording the optical density (OD) of 1 ml of vase solution taken from each treatment including control at 600 nm using PC-based UV–VIS spectrophotometer (Systronics) by the method of Naing et al. (2017) taking E. coli as standard (OD 1 = 8 × 108 cfu ml−1). The bacterial density was expressed as cfu ml−1. Solution uptake (ml) was evaluated as the difference between unutilized volume of solution remained at the completion of the experiment and total volume of solution in the vase. The timing of neck bending was determined by assessing the change in the position of the flower heads according to the method of Perik et al. (2012).

Membrane stability index

Electrolyte seepage provides a measure of membrane integrity of plant cells, which was calculated by incubating 500 mg petal tissue in 25 ml deionized water at 25 °C for 30 min and 95 °C for 15 min employing Sairam (1994) method. The conductivities of the samples incubated at 25 °C (C1) and those incubated at 95 °C (C2) were recorded by using Elico CM180 conductivity meter. MSI was evaluated by computing the formulae:

$${\text{MSI}} = \left[ {1 - {\text{C}}1/{\text{C}}2} \right] \times 100$$

Estimation of total phenols and sugar fractions

Quantification of phenols was performed by the method as described by Swain and Hillis (1959) using gallic acid as standard. A suitable volume of aliquot from the alcohol-soluble fraction of the tissue extract was diluted to 7 ml with distilled water, followed by the addition of 0.5 ml of Folin–Dennis reagent. After 3 min, 1 ml of saturated solution of sodium carbonate was added and the total volume was made to 10 ml with distilled water. Absorbance was measured after 30 min at 725 nm. Nelson’s (1944) protocol was employed for quantification of reducing sugars using glucose as standard. A suitable volume of aliquot from the alcohol-soluble fraction of the tissue extract was made up to 5 ml with distilled water, followed by the addition of 1 ml of copper reagent prepared by mixing copper reagent A and B in the ratio of 50:1. The mixture was heated at 100 °C for 20 min in water bath. After cooling at room temperature, 1 ml of arsenomolybdate reagent was added and the volume was made to 25 ml with distilled water. Absorbance was measured at 520 nm. Total sugars were estimated after enzyme mediated conversion of non-reducing sugars into reducing sugars by invertase. The volume of a suitable aliquot from the alcohol-soluble fraction of the fixed material was made to 4 ml with distilled water followed by the addition of 1 ml of 0.3% invertase. A drop of toluene was layered on the top and the solution was incubated overnight at 25 °C. Total sugars were then estimated by employing Nelson’s (1944) protocol. Non-reducing sugars were determined by evaluating the difference between total and reducing sugars. The parameters were expressed as mg g−1 fm.

Quantification of soluble proteins and α-amino acids

For quantification of soluble proteins, 1 g of petal tissue was macerated in 100 mM phosphate buffer (pH 7.2) comprising NaCl (150 mM), ethylenediamine tetraaceticacid (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 min. 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 following Rosen’s (1957) protocol using glycine as standard. The soluble protein and α-amino acid content was expressed as mg g−1 fm.

Determination of enzyme activities

Superoxide dismutase (SOD)

SOD activity was determined according to Dhindsa et al. (1981). One unit of SOD activity was defined as the quantity of enzyme reducing 50% absorbance of reaction mixture with enzyme in comparison to reaction mixture lacking enzyme. The reaction mixture contained sodium carbonate (50 mM), nitroblue tetrazolium (75 µM), ethylenediamine tetraacetic acid (0.1 M), methionine (13 mM) in 50 mM phosphate buffer (pH 7.8). The absorbance was monitored at 560 nm on spectrophotometer. The SOD activity was expressed as units min−1 mg−1 protein.

Catalase (CAT)

CAT activity was determined by employing Aebi (1984) protocol based on the consumption of hydrogen peroxide (H2O2) in the reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), enzyme extract (50 µl) and distilled water making a final volume of 3 ml. Absorbance of reaction mixture was recorded at 240 nm for three minutes using spectrophotometer. The activity was expressed as µM H2O2 red. min−1 mg−1 protein.

Ascorbate peroxidase (APX)

APX activity was assayed according to the protocol as described by Chen and Asada (1989) which is based on decrease in absorbance at 290 nm due to the oxidation of ascorbate (0.1 mM) in the reaction mixture. The reaction mixture in addition to ascorbate contained potassium phosphate buffer (50 mM) at neutral pH and H2O2 (0.3 mM). The absorbance was recorded at 290 nm for 3 min. The activity was expressed as µmol min−1 mg−1 protein.

Lipoxygenase activity (LOX)

LOX activity was determined by Axerold et al. (1981) method. To begin the reaction, 10 µl of petal extract was added to the mixture containing Tris–Hydrochloric acid buffer (50 mM) at pH 6.5 and linoleic acid (0.4 mM). The absorbance was recorded at 234 nm for 5 min and activity was expressed as µmol. min−1 mg−1 protein.

Statistical analysis

For determination of various parameters 3 replicates (each comprising two flowers) from each treatment were used except for 5 biological replicates used for assessment of vase life and floral diameter. Completely randomized experimental design was performed during the present study. Treatment means were compared by analysis of variance using SPSS (SPSS version 16; Chicago, USA). The significant difference among various treatments was analyzed by comparing treatments applying Duncan’s multiple range test (DMRT P < 0.05).

Results

Effect of SNP on flower longevity and floral diameter

Senescence commenced with the loss of turgidity in petals followed by upward erection and complete wilting of flowers (Fig. 1a–c). Flowers treated with SNP showed significant improvement in flower longevity. The recorded longevity of flowers treated with different concentrations of SNP viz., 50, 100, 150 and 200 µM was 8, 12, 10 and 4 days respectively. The flowers held in distilled water lasted for 6 days confirming that higher SNP concentration hastened the process of senescence. Moreover, the longer flower longevity was associated with larger floral heads compared to control which however showed a decreasing trend with the advancement of flower development from day 2 to 5 (Fig. 2a–b).

Fig. 1
figure 1

Effect of different concentrations of SNP on postharvest performance of isolated flowers of Calendula officinalis on day 0 (a), day 2 (b) and day 11 (c) of transfer of flower buds to the test solutions

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Fig. 2
figure 2

Effect of different concentrations of SNP on flower longevity (a) and floral diameter (b) of Calendula officinalis. Each value represents the mean of 5 replicates. Letters above the bars denote the statistical significance between individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Effect of SNP on bacterial density, solution uptake and neck bending

SNP was highly effective in inhibiting the microbial growth and neck bending in cut Calendula flowers. Least bacterial growth was recorded in vase solutions containing 100 µM SNP. The flowers held in this solution resulted in maximum solution uptake besides minimum neck bending in flower stems. Maximum bacterial density was recorded in the control which showed reduced solution uptake and faster rate of neck bending. Neck bending in untreated flowers was initiated on day 5, while as in flowers treated with various concentration of SNP viz., 50, 100, 150 and 200 µM, it was observed on day 7, 11, 9 and 3 respectively. With the advancement of flower development, a spike in bacterial density and rapid bent in flower stems was observed in flowers from day 2 to 5 of experiment. Since flowers treated with 200 µM SNP registered early wilting, so the bacterial density was determined on day 5 for the sake of convenience (Fig. 3a–c).

Fig. 3
figure 3

Effect of different concentrations of SNP on bacterial density (a), solution  uptake (b) and neck bending (c) in flowers of Calendula officinalis. Each value represents the mean of 3 replicates. Letters above the bars denote the statistical significance between individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Effect of SNP on membrane stability index (MSI)

As flower development progresses, membranes lose their integrity, leading to electrolyte leakage and loss of intracellular compartmentalization. However, SNP at 100 µM was highly effective in protecting the membrane deterioration by maintaining high membrane stability in petal tissues as compared to control. However, MSI values were observed as decreased as the flowers approached senescence from day 2 to 5 of the experiment. The observations pertaining to MSI are pictured in Fig. 4.

Fig. 4
figure 4

Effect of different concentrations of SNP on MSI in flowers of Calendula officinalis. Each value represents the mean of 3 replicates. Letters above the bars denote the statistical significance between the individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Effect of SNP on total phenols and sugar fractions

Flowers held in SNP showed higher content of sugar fractions and total phenols in comparison to untreated samples. 100 µM SNP was most effective in maintaining high content of these parameters followed by 150 µM SNP. The results also indicate the effectiveness of SNP in maintaining marginally higher content of reducing sugars than non-reducing sugars in the petal tissues. In addition, untreated floral tissues registered a sharp decrease in these parameters with the advancement of flower development compared to flowers treated with SNP at optimal concentration (Fig. 5a–d).

Fig. 5
figure 5

Effect of different concentrations of SNP on total phenols (a), total sugars (b), reducing sugars (c) and non-reducing sugars (d) in flowers of Calendula officinalis. Each value represents the mean of 3 replicates. Letters above the bars denote the statistical significance between the individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Effect of SNP on soluble proteins and α-amino acids

Flowers treated with SNP at 50, 100 and 150 µM recorded a significant increase in the amount of soluble proteins. Maximum protein enrichment was recorded in the petal tissues treated with 100 µM SNP. Pertinently, the samples which showed higher protein content recorded lower α-amino acids. Maximum α-amino acid content was recorded in the samples treated with 200 µM SNP followed by control. However, protein content decreased with the concomitant increase in α-amino acids as flower development progressed from day 2 to 5 as depicted in the Fig. 6a–b.

Fig. 6
figure 6

Effect of different concentrations of SNP on soluble proteins (a) and α-amino acids (b) in flowers of Calendula officinalis. Each value represents the mean of 3 replicates. Letters above the bars denote the statistical significance between individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Effect of SNP on activities of antioxidant and lipoxygenase enzymes

Application of SNP resulted in substantial augmentation in the activities of SOD, CAT and APX, besides maintaining an attenuated LOX activity in petal tissues. Maximum SOD activity was registered in flower petals treated with 150 µM SNP, while as 100 µM SNP was highly effective in maintaining elevated activities of APX and CAT enzymes. In addition, high LOX activity besides low SOD, CAT and APX activity was recorded in 200 µM SNP treated samples followed by petal tissues of untreated flowers. However, SOD, CAT and APX activities were observed as decreased at the later stages of experiment as the flowers approached senescence, besides a significant increase in LOX activity as depicted in Fig. 7a–d.

Fig. 7
figure 7

Effect of different concentrations of SNP on activities of SOD (a), CAT (b), APX (c) and LOX (d) in flowers of Calendula officinalis. Each value represents the mean of 3 replicates. Letters above the bars denote the statistical significance between individual treatments. Bars with different letters indicate significant differences by Duncan’s multiple range test (P < 0.05)

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Discussion

Since the realization that NO plays a key role in the regulation of cellular functions in plants, it has garnered a profound attention amongst researchers carrying investigations on postharvest physiology of cut flowers keeping in view its non-hazardous and eco-friendly nature. NO  has been revealed as an exceptional signaling molecule due to its versatile functions in physiology and biochemistry of living organisms (Lamattina et al. 2003; Astier et al. 2018). Improved longevity of Calendula flowers could be attributed to the involvement of NO in maitaining proper water conduction in the flowers by  inhibiting the bacterial growth in vase solutions that otherwise lead to neck bending (Perik et al. 2014). Maintenance of proper turgor pressure, achieved by sufficient water uptake and/or reduced rates of water loss in flower heads is a prerequisite to prevent head drop (Hemati et al. 2019). Additionally, NO could prevent neck bending by inducing lignification that has considerable scope in strengthening the flower pedicels (Böhm et al. 2010; Naing et al. 2017; Hemati et al. 2019). It has been observed that NO up-regulates the phenylalanine ammonia lyase (PAL) activity which is involved in synthesis of many phenolics and lignin like compounds associated with plant metabolism (Liu et al. 2005; Naing et al. 2017; Rezayian et al. 2020). So our analysis suggest that NO could prevent neck bending by inhibiting bacterial growth and xylem blockage, besides up-regulating the expression levels of lignin biosynthesis, gene and antioxidant activities. Furthermore, maintenance of proper water balance in petals resulted substantial improvement in head diameter of Calendula flowers consistent with the earlier findings by Zhang et al. (2018) and Deng et al. (2019) in cut lily and roses respectively.

Membrane stability plays a key role in preventing excessive leakage of electrolytes, sugars and pigments in order to delay senescence (Ezhilmathi et al. 2007; Ghadakchiasl et al. 2017). As the senescence advances, oxidative stress due to free radical production leads to deterioration of membranes hence loss of membrane integrity. NO could maintain high membrane stability in Calendula by inhibiting lipid peroxidation either directly by interacting with lipid peroxyl radicals or indirecty by maintaining a decreased LOX activity in the petal tissues (Shabanian et al. 2018; Singh et al. 2020). NO application has been reported to improve the vase life of various cut flowers such as carnations, gladiolus, chrysanthemums and lily by maintaining high membrane stability in the petal tissues. (Mansouri 2012; Naing et al. 2017; Kazemzadeh-Beneh et al. 2018; Zhang et al. 2018).

The role of NO to confront senescence in Calendula flowers might be related with its regulatory role in maintaining higher sugar levels in the petal tissues. Limited information is available about the mechanism involved in NO induced carbohydrate metabolism in cut flowers. Our observations suggest that NO maintained high sugar content in petals possibly by modulating the enzyme activities of carbohydrate metabolism and/or by acting as a potent inhibitor of the mitochondrial respiratory chain which prevented rapid degradation of sugars (Mason et al. 2006; Han et al., 2018). Sugars play an indispensable role in preventing senescence not only by acting as typical osmo-protectants and membrane stabilizers (Couée et al. 2006) but also by eliciting signals  during sugar sensing and signaling systems (Chen et al. 2009). Sugar molecules have been shown to stabilize the membrane lipids via hydrogen-bonding interactions between sugar OH and lipid head-groups. Such interactions are beneficial in ROS balancing and preventing the phase transitions that otherwise elicit extravasation of solutes (Vital et al. 2019). Furthermore, petal senescence occurs concomitant with rise in ratio of non-reducing to reducing sugars (Nichols 1973). However, NO maintained higher ratio of reducing to non-reducing sugars in petal tissues of Calendula flowers. NO has reportedly increased sugar content in petal tissues of many cut flowers such as chrysanthemums and gerbera due to which the longevity in these flowers was significantly enhanced (Mansouri 2012; Hemati et al. 2019).

Petal browning and discoloration are important parameters which determine display quality and market value of cut flowers and in many cases are responsible for the termination of vase life (Khalaj et al. 2017; Salehi Salmi et al. 2018). Flowers treated with NO recorded high phenolic content in petal tissues due to increased PAL activity as discussed above, which is responsible for the synthesis of several defense-related secondary compounds that prevent cell membranes from lipid peroxidation by scavenging free superoxide radicals (Soleimani Aghdam et al. 2015; Naing et al. 2017; Shabanian et al. 2018) consistent with the earlier observations by Naing et al. (2017) in carnations.

Higher levels of soluble proteins in SNP treated petal tissues could be owed to the involvement of NO in up-regulation of m-RNA transcription and protein synthesis (Beligni and Lamattina 2001; Zhang 2007). Moreover, NO might have inhibited the activity of proteases by down-regulating their gene expression, thus reducing the α-amino acid content in the petal tissues (Pak and Doorn 2005; Dwivedi et al. 2016). Furthermore, the accumulation of proteins may be involved in enhancing the activities of antioxidant enzymes and synthesis of specific stress-related proteins as defense mechanism (Doganlar et al. 2010; Promyou et al. 2012). Noticeable accumulation of total soluble proteins by NO treatment might alternatively amplify tolerance of cells through osmotic regulation synergistically with sugars. Studies also suggest that protein enrichment minimizes the starving effect of carbohydrates/sugars by serving as alternative respiratory substrates (Rezvanypour and Osfoori 2011). Pertinently, NO has been shown to maintain an elevated protein levels in lilium, gladiolus, gerbera and white prosperity flowers improving their longevity (Kaviani et al. 2013; Dwivedi et al. 2016; Shabanian et al. 2018; Kazemzadeh-Beneh et al. 2018).

Antioxidant system is regarded as one of the most efficient systems to counter deleterious effects of ROS associated oxidative stress (Soares et al. 2019). It comprises of both enzyme and non-enzyme players which function synergistically to protect cells from oxidative imbalances and stress (Hossain et al. 2015; Hoque et al. 2016; Soares et al. 2019). NO has been found to act as an antioxidant molecule either directly by scavenging the ROS or indirectly by modulating other antioxidant systems such as antioxidant enzymes to diminish the obnoxious effects of both biotic and abiotic stresses in plants (Zeng et al. 2011; Ahmad et al. 2016; Dwivedi et al. 2016; AbuQamar et al. 2017; Siddiqui et al. 2017; Deng et al. 2019; Hemati et al. 2019; Rabiei et al., 2019; Sharma et al. 2020; Souri et al. 2020). In the present investigation, NO positively amplified the activities of antioxidant enzymes possibly by up-regulating the gene expression and protein functions (Nabi et al. 2019; Sharma et al. 2020). Validating our results, NO has improved the vase life of gladiolus, gerbera, carnations and rose by modulating the antioxidant enzymes (Dwivedi et al. 2016; Naing et al. 2017; Kazemzadeh-Beneh et al. 2018; Deng et al. 2019). Furthermore, our result has been consistent with the observations of Dwivedi et al. (2016) which stated that in addition to increased activities of SOD, CAT and APX, an attenuated LOX activity was recorded in SNP treated gladiolus florets/spikes.

Conclusion and future outlook

The present investigation reveals promising effect of NO on maintaining the postharvest quality of Calendula flowers. Among the range of treatments, 100 µM SNP was observed as most effective in improving the postharvest performance and longevity in this flower followed by 150 µM SNP. The important finding of this investigation was that NO through SNP, delayed neck bending in Calendula by inhibiting bacterial growth and xylem blockage, besides promoting accumulation of phenolic compounds. The delayed neck bending was corroborated with the up-regulated activities of antioxidant enzymes, high membrane stability, in addition to an elevated sugar content and protein levels in petals of Calendula officinalis. However, critical revision needs to be undertaken at molecular level to explain how exactly NO affects various parameters during senescence in cut Calendula flowers. The authors realize that Calendula officinalis could act as a model system for studies related to postharvest physiology and petal cell death.