Senescence in plants is regulated by interplay between various growth regulators like ethylene, gibberellins, cytokinins, abscisic acid and auxins. Ethylene is the main  candidate for senescence in ethylene sensitive flowers, while as it has little or no role to play in ethylene insensitive flowers (van Doorn and Woltering 2008). In ethylene dependent flower systems such as carnation, ethylene antagonists like amino oxyacetic acid (AOA), silver thiosulfate (STS) and amino vinyl glycine (AVG) have been shown to delay flower senescence (Satoh et al. 2005). Moreover, ethylene biosynthesis competitor, polyamines have been reported to show anti-senescence properties in ethylene dependent flower systems (Galston 1983; Sawhney et al. 2003). Polyamines delay senescence by retarding membrane deterioration, RNase and protease activity (Sawhney et al. 2003). The most abundant polyamines present in plants are putrescine, spermidine and spermine.  The  interplay between polyamines and ethylene appears to regulate flower development and senescence. Although, polyamines and ethylene share the common precursor, S-adenosyl methionine (SAM), but ethylene accelerates senescence, whereas polyamines tend to have delaying effect (Sawhney et al. 2003). The antagonistic roles of polyamines and ethylene can be ascribed to their competition for the common precursor, SAM (Galston 1983).  The role of exogenously applied polyamines still remains elusive. The present study was persued to assess the effect of polyamines (putrescine, spermidine and spermine) on flower development and senescence in N. plumbaginifolia  which follows a well defined senescence pattern, that can be physiologically monitored and documented.

Isolated buds of N. plumbaginifolia at brush stage, i.e., 1 day before anthesis (stage IV) were used for the present study (Figs. 1, 2). The pedicels (1 cm) were divided into three sets and held in 5 ml glass vials containing 3 ml of respective holding solutions. Each set was supplied with different polyamines, viz., putrescine, spermidine and spermine. The concentration of all the vase solutions was kept 10 mM after standardization of the optimal concentration in all the cases. A separate set of 35 buds held in distilled water served as control. The treatment effects were evaluated by keeping the experiment under natural conditions in the laboratory with temperature of 21 ± 2 °C under cool white fluorescent light with a mix of diffused natural light (10 W m−2) for 12 h a day and relative humidity (RH) of 60 ± 10 %. The day of transfer of buds to distilled water (DW) was designated as day zero (D0). Floral diameter was recorded on day 2 and day 4 of transfer to the vase solutions. Total soluble proteins, α-amino-acids, phenols and sugar fractions (reducing, non-reducing and total sugars) were estimated on day 3 of the transfer.

Fig. 1
figure 1

Flowers of Nicotiana plumbaginifolia L. in full bloom in the month of July in Kashmir University Botanic Garden

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Fig. 2
figure 2

Stages of flower development and senescence in Nicotiana plumbaginifolia L. The floral buds at stage IV (1 day before anthesis) were collected for experimentation

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Proteins were estimated by the method of Lowry et al. (1951) using BSA as the standard. Reducing sugars were estimated by method of Nelson (1944) using d-glucose as the standard. Total sugars were estimated by enzymatic conversion of non-reducing sugars to reducing sugars. Non-reducing sugars were calculated as the difference between total and reducing sugars. α-amino acids were estimated by the method of Rosen (1957) using glycine as the standard. Total phenols were estimated by the method of Swain and Hillis (1959) using gallic acid as the standard. Experiment was conducted under complete randomized design. The data was analyzed statistically and LSD was computed at P0.05 using SPSS 16 software.

Treatment of isolated buds of N. plumbaginifolia with different polyamines resulted in the improvement of its postharvest attributes. Maximum flower longevity was registered in the flower buds held in spermine, which showed an  improvement in flower longevity by 3 days, followed by 2 days in putrescine and 1 day in spermidine (Fig. 3; Table 1). Earlier studies have shown that polyamines delay senescence in flower like Dianthus and Narcissus, but spermidine was found to be effective than putrescine and spermine (Lee et al. 1997; Sardoei et al. 2013). Delay in flower senescence by the application of polyamines can be ascribed to its suppression of ethylene biosynthesis and action (Sardoei et al. 2013).

Fig. 3
figure 3

Effect of putrescine spermidine and spermine on flower longevity in isolated flowers of Nicotiana plumbaginifolia L. on 0 (A), 2 (B) and 6 day (C) of transfer to the respective test solutions

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Table 1 Effect of various polyamines (putrescine, spermidine and spermine) on flower longevity, floral diameter, soluble proteins, total amino acids, total phenols, total sugars, reducing sugars and non-reducing sugars on isolated flowers of Nicotiana plumbaginifolia L.
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The isolated flowers supplied with putrescine, spermidine and spermine in their test solutions maintained higher floral diameter than the control. Highest floral diameter was registered in the flowers held in spermine on day 2 of transfer to the test solution, followed by putrescine and spermidine (Table 1). The increase in floral diameter in the samples treated with polyamines can be attributed to greater water uptake and thereby turgidity of flowers, because of the maintenance of higher carbohydrates in the petal tissues (Dar et al. 2014). Increase in content of carbohydrates in petals  may cause a  more negative water potential in the flowers, which will ultimately result in increased water uptake, and thereby increased floral diameter (van Doorn 2004). The floral buds treated with putrescine, spermidine and spermine showed  considerably higher values for soluble proteins and α-amino acids (Table 1). The floral buds held in spermine showed highest soluble protein and α-amino acid content as compared to other treatments including control. Similar results of increase in protein content by the application of polyamines have been reported in flowers like Dianthus and Gladiolus (Tassoni et al. 2006; Nahed et al. 2009). Polyamines are thought to delay flower senescence either by minimizing ethylene production or by stabilizing proteins. Polyamines bind to the carboxylic (COOH) group of the amino acids (proteins) and prevent them from the action  by free radicals and other proteolytic enzymes (Galston 1983). Polyamines have also been found to delay protein degradation in various flowers  such as carnation (Galston and Sawhney 1990). Protein degradation is considered to be the earliest step in the senescence of various flowers like Petunia, Rosa, Iris, Dianthus, Hemerocallis, Sandersonia and Gladiolus (Tripathi et al. 2009). The application of polyamines to the floral buds of N. plumbaginifolia resulted in decrease in total phenolic content (Table 1). The floral buds held in spermine showed lowest phenolic content. The increased phenolic content associated with senescent flowers corroborates the earlier findings on various flowers like Iris and Dianthus (Dar et al. 2014; Ahmad and Tahir 2015).

A marginal increase in sugar fractions (total, reducing and non-reducing) was registered in the floral buds treated with different polyamines.  Maximum values for sugar fractions were  recorded in the floral buds held in putrescine, followed by spermine and spermidine (Table 1). Our results are in agreement with the earlier work on Gladiolus (Nahed et al. 2009).  The  increase in the carbohydrate content due to the application of polyamines can be attributed to the regulated consumption of the storage carbohydrates through respiration by delaying the activity of ethylene (Galston and Sawhney 1990). Studies on various flower systems like Iris and Dianthus have suggested that increased carbohydrate levels are associated with increased floral longevity (Ahmad et al. 2013; Dar et al.2014). Moreover, exogenous application of sugars have been found to delay the rise in ethylene production mainly by shifting the polyamine-ethylene balance towards polyamines in various ethylene sensitive flower systems, thereby improving their postharvest performance (van Doorn 2004).

Our study reveals that the polyamines increase flower longevity by maintaining higher protein and carbohydrate content in the petal tissues. Among the polyamines tested, spermine was found to be the most effective in delaying flower senescence and maintaining  the postharvest quality of N. plumbaginifolia.