Vase life, ethylene production, and relative fresh weight (RFW)
The vase life of cut carnation flowers (Tico Viola) responded differently to SNP, and the effects were dose-dependent (Additional file 1: Figure S1). Based on the results shown in Fig. 1a, most of the treatment concentrations extended the vase life compared with the control, whereas the vase life of flowers treated with 20 mg L−1 SNP declined. Specifically, at 10 mg L−1, SNP significantly extended the vase life by approximately 6 days, and there was an increase of 2.6, 3.8, and 1.6 days at SNP concentrations of 1, 5, and 15 mg L−1, respectively, compared with the control. Thus, 10 mg L−1 was considered the optimal SNP concentration and used for further experiments.
Generally, the vase life of cut flowers was associated with their ethylene production during the vase life period. For the control and SNP-treated (1, 15, 20 mg L−1) cut flowers, the ethylene production rate was relatively low at day 3; however, it began to periodically increase and tended to reach a peak by day 9 (Fig. 1b). In fact, the ethylene production rate of the flowers at day 9 after the treatment was observed to be the highest in control and 20 mg L−1 SNP treatments, showing petal in-rolling (sign of senescence) on days 9 and 8 after the treatment, followed by ethylene production in SNP treatments at 15 and 1 mg L−1, respectively. In case of other SNP treatments (5 and 10 mg L−1), the ethylene production rate increased markedly on day 9 and further increments were noticed until day 12 (data not shown); however, the ethylene production rate in 10 mg L−1 SNP treatment seemed to increase until day 15, when the flowers finally showed petal in-rolling. Overall, the ethylene production rate was likely to be associated with the symptoms of flower senescence such as petal in-rolling and wilting.
Normally, RFW is also strongly associated with the vase life of cut flowers. In this study, the changes in RFW of cut carnation flowers exhibited similar trends in both control and SNP treatments. RFWs were the highest on day 3 for the control and some of the SNP treatments (1, 15, and 20 mg L−1), and decreased thereafter (Fig. 1c). Similarly, 5 and 10 mg L−1 SNP treatments also gave the highest RFWs on day 3; whereas the RFWs of 10 mg L−1 SNP were not significantly different between days 3 and 6, but they declined thereafter, in fact, the RFW of 10 mg L−1 SNP treatment on day 9 was still higher than that of 5 mg L−1 SNP treatment (Fig. 1c). Throughout the vase life period, the RFWs of 10 mg L−1 SNP treatment were significantly higher than that of the other treatments.
Quantification of the genes related to ethylene production and flower senescence
To understand the relationship between ethylene production and the expression level of ethylene biosynthesis or receptor genes, the transcript levels of the ethylene biosynthesis (DcACS1 and DcACO1) genes were determined on day 9. The expression profiles of the ethylene biosynthesis genes are presented in Fig. 2a and b. As expected, the transcript levels of the detected genes expressed in the flowers on day 9 were the highest in the control and 20 mg L−1 SNP treatment, followed by other SNP treatments (15, 1 and 5 mg L−1), whereas the lowest expression levels were noted in the 10 mg L−1 SNP treatment. These findings support the conclusion that ethylene production was strongly associated with ethylene-related gene expression.
Unlike the ethylene production-related genes, the expression and transcript levels of DcCPI were observed to be the highest in the 10 mg L−1 SNP treatment, followed by the 5, 15, and 1 mg L−1 treatments, whereas the lowest levels were observed in the control and 20 mg L−1 SNP treatments (Fig. 2c). These findings indicated that DcCPI plays important role in the petal senescence of cut carnation flowers.
Antioxidant activity
In the Tico Viola carnations, petal senescence in the control flowers was observed on day 9, whereas SNP treatments extended the vase life of the flowers. Thus, on day 9, we determined the ROS-scavenging activity using DPPH and ABTS assays, and the total polyphenol and total flavonoid content of the flowers. The ROS-scavenging activity in all SNP treatments except 20 mg L−1 was significantly higher than in the control (Fig. 3a and b). Specifically, the activity was the highest in 10 mg L−1 SNP treatment followed by the other SNP treatments (5 > 1 > 15 mg L−1). Additionally, the antioxidant activity profiles (for total polyphenol and flavonoid) for both the control and the SNP treatments were also similar to those of ROS-scavenging activity (Fig. 3c and d). From the findings, it is obvious that SNP extends vase life of carnations by enhancing the antioxidant activity and reducing the transcript levels of the genes involved in ethylene production and petal-senescence.
Treatment with ACC
It was of interest to confirm the effect of SNP treatment on the vase life of cut flowers in combination with an ethylene precursor (ACC). When ACC was added to the vase solution (distilled water), flowers showed obvious signs of senescence (petal in-rolling) by day 7 (Additional file 1: Figure S2) and the vase life was found to be 2 days shorter than that of the controls; however, addition of ACC to the vase solution containing 10 mg L−1 SNP extended the vase life by 1.3 days over the controls (Fig. 4a). Although ACC inhibited the vase life of the flowers, when it was combined with 10 mg L−1 SNP, the vase life of the flowers was still longer than that in the control. Thus, this result supports a positive effect of SNP on the vase life of cut carnation flowers.
Moreover, the ethylene production rate in the flowers treated with ACC alone rapidly increased and was higher than that in control, particularly on days 6 and 9, whereas the production on day 6 between control and the combination (ACC and SNP) was not significantly different (Fig. 4b). However, the ethylene production rapidly increased on day 9 and vase life of the flowers also ended after day 10. In addition, the RFW obtained for ACC was also lower than control on days 6 and 9 (Fig. 4c). When ACC was added to the SNP-containing solution, a distinct increase in RFW was noted.
In response to ACC addition, the vase life of the cut flowers was shortened by increased ethylene production. Thus, the transcript levels of the ethylene production-related genes and the petal senescence-related genes were determined in the ACC-treated flowers along with controls. As expected, the transcript levels of DcACO1 and DcACS1 genes were significantly higher in ACC treatment than in controls, whose transcript levels were, in turn, higher than those in the ACC + SNP treatment (Fig. 5a and b). In addition, the transcript level of the petal senescence-related gene (DcCPI) expressed in flowers treated with ACC + SNP was also higher than in both controls and flowers treated with ACC, whereas the transcript levels were higher in controls than in ACC treatment (Fig. 5c).
Moreover, we also determined the ROS-scavenging activity (DPPH and ABTS activity) and antioxidant activity (total polyphenol and flavonoid content) in the flowers treated with ACC, to examine whether the activity was reduced earlier than in the controls. Results shown in Fig. 6 indicate that the ROS-scavenging activity in the ACC treatment was lower than that in controls; however, the activity increased when SNP was combined with ACC. Similarly, the total polyphenol and flavonoid content detected in ACC was also lower than that in controls, but SNP addition enhanced this activity. These findings suggest that SNP has the ability to extend the vase life of flowers even when present in combination with ACC, by increasing the antioxidant activity, and by reducing the transcript levels of ethylene production-related and petal senescence-related genes.
Effect of SNP on vase life of different genotypes
We designed experiments to confirm the role of this SNP concentration (10 mg L−1) in different carnation cultivars, i.e., Venus, Tico Tico, and Shino Lily. As shown in Fig. 7a, the length of the flower vase life did not differ significantly among the tested cultivars; however, the vase life of Venus was 9 days, one day longer than that of the other two cultivars. When 10 mg L−1 SNP was used, the vase life of all cultivars improved. The vase life was 15 days for Venus and 12 days for Tico Tico and Shino Lily, resulting in an improvement of 4 days for the cultivars Tico Tico and Shino Lily, and 6 days for Venus, compared with the respective controls (Fig. 7a and Additional file 1: Figure S3). In addition, ethylene production by all cultivars was significantly higher in the controls than in SNP treatments (Fig. 7b); ethylene production for controls started increasing markedly on day 6 and reached a peak by day 9, when most of the vase lives ended. Thus, we predicted that the peak of ethylene production rate for treatments would be within days 12–15 because the cultivars’ vase lives ended around this period. RFWs for all the cultivars reached the highest values during the first 3 days after treatment, for both controls and treatments, and it declined thereafter; a quick decrease was observed in the controls, whereas a slow decrease was noted in the treatments (Fig. 7c).
In addition, as expected, the transcript levels of the ethylene biosynthesis genes (DcACO1 and DcACS1) were also significantly higher in controls than in SNP treatments for all the cultivars (Fig. 8). Furthermore, the transcript levels of the senescence-related gene (DcCPI) were also higher in the SNP treatments than in the controls (Fig. 9).
Significantly higher ROS-scavenging activity and antioxidant activity were also detected in the SNP treatments than in the controls, for all cultivars (Fig. 10). Therefore, the findings of the genotype experiment also lent strong support the conclusion that SNP extended the vase life of cut carnation flowers by improving all the parameters that are associated with vase life of the flowers.