“From villain to hero”: Harnessing the gaseous grace of nitric oxide for prolonged elegance in Antirrhinum majus L. cut spikes

2.1 Experimental setup

Uniform and healthy spikes of Antirrhinum majus from the experimental plots situated within the Kashmir University Botanical Garden (KUBG) were collected during the morning hours. These selected spikes were characterized by having the most mature bud at one day before anthesis stage (stage III). To preserve their freshness, the spikes were promptly immersed in distilled water and transported to the laboratory. Each spike was then trimmed to a standardized length of 30 cm to ensure uniform spike length. Subsequently, the spikes were divided into four sets, with each set comprising 10 flasks, and two spikes were placed within each flask. Test solutions of Sodium nitroprusside (SNP) were prepared carefully, with concentrations of 10 μM, 20 μM and 30 μM following rigorous standardization protocols. A control group consisting of 10 flasks with spikes immersed in distilled water was established to serve as a reference for comparative analysis. The first three sets of spikes were pulsed with 10, 20 and 30 μM of SNP for 24 h and then these pulsed spikes were transferred to distilled water containing flasks. The experiment was conducted under controlled conditions, encompassing a relative humidity of 60 ± 10 %, a consistent 12- hour light cycle and an average temperature of 23 ± 2 °C. The day on which the spikes were transferred to the test solutions was designated as day zero (D0). On day 2 and 6, comprehensive evaluations were conducted, encompassing a wide array of parameters, including vaselife, floral diameter, membrane stability index, soluble proteins, total phenols, sugar fractions, membrane stability index, soluble proteins, total phenols, sugar fractions (total, reducing and non-reducing), solution uptake, bacterial density, ethylene content, catalase activity, ascorbate peroxidase activity, superoxide dismutase activity and lipoxygenase activity.

2.2 Vaselife and floral diameter

The evaluation of the vase life of cut A. majus spikes involved visual monitoring of senescence indicators such as wilting and tepal abscission. The average vase life was determined by recording the number of days from the transfer of the spike until 70 % of the florets exhibited senescence on each spike. Additionally, floral diameter measurements were taken on day 2 and day 6 post-spike transfer, with the measurement involving the calculation of the average of two perpendicular distances across a flower.

2.3 Bacterial density and solution uptake

The assessment of bacterial density was carried out by measuring the optical density of 1 ml of solution from each treatment including control at 600 nm using spectrophotometer. The E. coli was taken as reference organism (where 1 OD was equivalent to 1x10⁸ CFU/ml). Solution uptake was determined by calculating the difference between the volume of the vase solution at the end of the experiment and the initial volume of the vase solution (Naing et al., 2017b).

2.4 Ethylene measurement

Ethylene was quantified by employing Whitehead et al. (1992) protocol. The flower petals at different developmental stages of A.majus were collected and kept in 3 glass vials sealed tightly with parafilm. After a 2 h incubation, 1 ml of the gaseous mixture was extracted from each vial and injected into gas chromatograph (Nucon model 5700) fitted with a flame ionization detector. The concentration of ethylene was assessed using a calibration curve derived from known ethylene standard.

2.5 Membrane stability index (MSI)

The assessment of Membrane Stability Index (MSI) involved measuring solute leakage from petal tissues. This was determined by incubating 100 mg of petal tissue in 5 ml of deionized water at 25 °C for 30 min and subsequently at 100 °C for 15 min, following the methodology described by Sairam, 1994. The MSI was calculated using the formula: $$\mathit{MSI}=\left(1-C1/C2\right)\ast 100$$ Here, C1 denotes the conductivity of the samples after incubation at 25 °C, and C2 denotes the conductivity at 100 °C. The conductivity values were recorded using the Elico CM180 Conductivity meter.

2.6 Quantification of soluble proteins

To estimate protein content, 1 g of petal tissue was crushed in a 100 mM phosphate buffer with a pH of 7.2, comprising 10 % polyvinyl pyrrolidone (PVP), 1 mM EDTA, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, and 1 mM Dithiothreitol (DTT). The resulting mixture underwent centrifugation in a refrigerated centrifuge at 12,000xg and 5 °C for 15 min. Protein estimation was carried out using a portion of the collected supernatant, following the method outlined by Lowry et al., 1951.

2.7 Quantification of phenols and sugars

To determine sugar fractions and phenols, 1 g of chopped petal tissue from each treatment was immersed in hot 70 % ethanol, macerated, and subjected to three rounds of centrifugation. From an appropriate portion of the resulting supernatant, total phenols, reducing sugars, non-reducing sugars, and total sugars were assessed. The quantification of total phenolics followed the method outlined by Swain and Hillis in 1959, utilizing gallic acid as a standard. Nelson's method from 1944 was applied to estimate reducing sugars, with glucose serving as the standard. To determine total sugars, non-reducing sugars were converted to reducing sugars using invertase. The quantity of non-reducing sugars was calculated as the difference between total and reducing sugars.

2.8 Extraction and assessment of enzyme Activity

2.9 Experimental design and statistical analysis

The completely randomized experimental design was followed during the study and statistical significance between individual treatments was determined through Duncan’s test at P < 0.05 and each value represents a mean of three replicates.

3.1 Vaselife

Flower senescence in A. majus was characterized by incomplete flower opening, pigmentation suppression, spike bending and ultimately petal abscission. The introduction of sodium nitroprusside (SNP) into the preservative solutions led to a remarkable extension in the vaselife of the excised spikes in comparison to the control as shown in Fig. 1. The application of SNP demonstrated a significant enhancement in vaselife, with the most substantial improvement observed at 20 µM. Nevertheless, a gradual reduction in longevity was observed beyond this concentration. The treated spikes exhibited a lifespan of 11, 18 and 13 days for 10 µM, 20 µM and 30 µM SNP respectively. It is noteworthy that all SNP treatments significantly prolonged vaselife compared to the control, having vaselife of only 8 days (Fig. 2A).

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Fig. 1

Photograph showing the effect of various standardized concentrations of SNP on postharvest longevity in cut pikes of A. majus.

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Fig. 2

(A-C): Graphs illustrating the impact of varying concentrations of SNP on the duration of postharvest longevity, floral diameter and content of soluble proteins in the floral tissues of A. majus cut spikes.

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3.2 Floral diameter

Regular assessments conducted on day 2 revealed a notable expansion in floral diameter among the groups treated with sodium nitroprusside (SNP), particularly at a concentration of 20 µM, in contrast to the control group. However, as the experiment progressed towards day 6, a significant reduction in floral diameter became apparent, with the control displaying the most pronounced decrease compared to the flowers treated with SNP (Fig. 2B).

3.3 Soluble proteins

In our study, soluble protein content in petal tissues was assessed following exposure to various concentrations of sodium nitroprusside (SNP) at regular intervals. Initially, an elevation in SNP concentration corresponded to an upsurge in protein content, reaching its peak at 20 µM SNP. However, as the SNP concentration surpassed this threshold, there was a subsequent decline in soluble protein content. On day 6, protein content decreased across all treatment groups, regardless of the specific treatment applied. The control group exhibited the most significant decrease, whereas the spikes treated with SNP test solutions showed a relatively smaller reduction in soluble protein content (Fig. 2C).

3.4 Total phenols

The cut spikes immersed in sodium nitroprusside (SNP) test solutions exhibited a conspicuous enrichment of phenolic compounds in their petal tissues when compared to the control. The most substantial surge in phenolic concentration was discerned in the floral buds treated with 20 µM SNP, followed by those treated with 30 µM SNP surpassing the phenolic content of other tested concentrations as well as the control group. Significantly, phenolic content displayed a temporal decline as time advanced, with the most pronounced decrease observed in the untreated spikes (Fig. 3A).

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Fig. 3

(A-D): Graphs illustrating the impact of varying concentrations of SNP on (A) phenols, (B) total sugars (C) reducing sugars and (D) non-reducing sugars in the floral tissues of cut spikes of A. majus.

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3.5 Sugars fractions (Total, reducing and non-reducing)

The application of sodium nitroprusside (SNP) treatments caused considerable augmentation in the content of total, reducing and non-reducing sugars compared to the control. Intriguingly, on day 2 the cut spikes subjected to 20 µM SNP treatments displayed the highest concentrations of total, reducing and non-reducing sugar components. These findings underscore the overall elevation in sugar content within the petal tissues of floral buds treated with SNP. Nevertheless, as the experiment progressed to day 6, a declining trend in the sugar fractions was witnessed across all treatment groups, including the control Fig. 3 (B-D).

3.6 Membrane stability index (MSI)

The membrane stability index (MSI) of the excised spikes experienced a remarkable enhancement when subjected to sodium nitroprusside (SNP) treatments, surpassing the MSI values of the control group. The most pronounced augmentation in MSI was evident at concentrations of 20 µM and 30 µM SNP, signifying the notable influence of SNP treatments on preserving membrane integrity. However, at day 6, a steep descent in MSI values was observed across all sets of treatments under observation, with the most substantial decrease observed in the untreated spikes (Fig. 4A).

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Fig. 4

(A-D): Graphs illustrating the impact of varying concentrations of SNP on (A) membrane stability index, (B) bacterial density, (C) solution uptake, and (D) ethylene content in the petal tissues of cut spikes of A. majus.

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3.7 Bacterial density and solution uptake

The utilization of various concentrations of sodium nitroprusside (SNP) exerted a dual impact on both solution uptake and bacterial density, establishing a unique interplay between these two factors. The pulsing of SNP led to a reduction in bacterial density when compared to the control group, ultimately resulting in an augmented uptake of the solution. Specifically, the concentration of 20 µM SNP coincided with the lowest bacterial density, concomitant with the highest degree of solution uptake. However, as the experiment proceeded to day 6, all treatments including the control group experienced a substantial elevation in bacterial density, coinciding with the lowest level of solution uptake (Fig. 4B,4C).

3.8 Ethylene content

The incorporation of sodium nitroprusside (SNP) treatments led to a conspicuous attenuation of ethylene production within the excised spikes in comparison to the control. The most substantial decline in ethylene synthesis was witnessed in the spikes exposed to 20 µM SNP. Nonetheless, as the experiment advanced, an escalation in ethylene production was observed across all treatment groups including the control group, signifying a diminishing efficacy of SNP over time (Fig. 4D).

3.9 Catalase (CAT) activity

The ongoing investigation unveiled a noteworthy augmentation in catalase activity within the floral buds of the excised spikes treated with sodium nitroprusside (SNP) in comparison to those treated with distilled water. Specifically, the highest catalase activity was detected in the floral buds exposed to 20 µM SNP, surpassing the activity levels of other concentrations as well as the control group. However, as the study progressed, a gradual descent in catalase activity was observed in all experimental groups, including the control, indicating a diminishing trend in catalase function over time (Fig. 5A).

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Fig. 5

(A-D): Graphs illustrating the impact of varying concentrations of SNP on the (A) catalase, (B) ascorbate peroxidase, (C) superoxide dismutase and (D) lipoxygenase activity in the petal tissues of cut spikes of Antirrhinum majus.

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3.10 Ascorbate peroxidase (APX) activity

The application of sodium nitroprusside (SNP) treatments resulted in a significant augmentation APX activity in the petal tissues. The APX activity increased with the application of SNP, reaching its zenith at 20 µM and beyond this concentration, a precipitous decline in APX activity became apparent. As the study progressed, there was an overall decline in APX activity in all the treatments and the control, signifying a reversal of the initial increase and indicating a decline in APX activity over time (Fig. 5B).

3.11 Superoxide dismutase (SOD) activity

The application of sodium nitroprusside (SNP) treatments led to a gradual augmentation in superoxide dismutase (SOD) activity within the cut spikes compared to the untreated ones. Moreover, the spikes treated with a lower concentration of SNP (20 µM) exhibited the highest SOD activity among the various SNP treatments employed. However, as the study progressed, a decline in activity of SOD was noted across all treatment groups, with the untreated spikes experiencing the most significant decrease (Fig. 5C).

3.12 Lipoxygenase (LOX) activity

The utilization of sodium nitroprusside (SNP) exerted a pronounced influence on the lipoxygenase (LOX) activity of the excised spikes. These findings showcased a substantial reduction in LOX activity following the administration of SNP treatments, particularly at a concentration of 20 µM SNP, where the lowest levels of activity were noted. Furthermore, irrespective of the specific treatment, there was a consistent upward trajectory in LOX activity with the passage of time across all experimental groups (Fig. 5D).