Number and length of adventitious root as affected by BR concentrations
In order to investigate the effect of different concentrations of BR on adventitious roots, the cucumber explants were exposed to different levels of BR solutions (0, 0.2, 1, 2, 4 and 8 μ M). The root length and root number of adventitious roots initially increased and then decreased with the increase of BR concentration, and both reached the maximum values at 1μM level (Fig. 1). Thus, the optimum concentration of BR (1 μM) was used in the subsequent experiment.
Number and length of adventitious root as affected by NO scavenger and inhibitors
The effect of NO scavenger (c-PTIO), NOS-like enzyme inhibitor (L-NAME) and NR inhibitor (Tungstate) on BR -induce adventitious rooting was investigated. As shown in Fig. 2, compared with BR treatment, 200 μM c-PTIO, 20 μM L-NAME or 100 μM Tungstate applied in combination with BR treatment significantly inhibited adventitious root formation. The adventitious root number and length of explants treated with SNAP (NO donor) plus BR were significantly higher than those of explants treated with either BR or SNAP alone.
Time course effects of endogenous NO content, NOS-like and NR activity
The time-course of NO content affected by 1 μM BR or 1.5 μM BRZ treatment was shown in Fig. 3A. Compared with the control, the NO content of BR-treated explants has a slow downward trend at 0h-6 h, this may be due to the wound response. From 6h to 24 h, the NO content of BR-treated explants increased, subsequently, a gradual decrease appeared until 48 h (Fig. 3A). The content of NO in the BR treatment reached the maximum at 24 h and was about 1.8 times as compared to the control. In addition, the NO content of BRz –treated explants gradually decreased from 0h to 48h, and the NO levels was always lower than those of BR-treated explant. Thus, the data suggested that BR regulated the level of endogenous NO in promoting the development of cucumber adventitious roots.
To explore the relationship between BR and NO, we further analyzed the effects of BR on the activities of NOS-like and NR enzymes in cucumber explants during rooting formation (Figs. 3B and 3C). The application of BR distinctly affected the time course of NOS-like activity. The NOS-like activity in BR-treated explants decreased slightly at 0-6 h, and then increased rapidly from 6h to 24 h, and reached the maximum at 24h, which was about 2 -fold of the control. Then, from 24h to 48h, the trend of NOS-like activity changed to gradual decrease (Fig. 3B). Meanwhile, compared with the control and the BR treatment, the NOS-like enzyme activity of BRZ treatment continuously decreased throughout the whole progress (Fig. 3B). It was suggested that the inhibition of NOS-like enzyme activity by BRz was very obvious. Similarly, the NR activity of explants treated with BR decreased transiently during the first 6 h, followed by a significant increase from 6h to 24 h, which reached its highest activities at 24 h (about 1.4-fold of the control), and then decreased at 48 h (Fig.3C). However, the NR activity of BRz treated explants continuously decreased during the range of 0-48 h (Fig.3C). In conclusion, the activity of the NOS-like and NR enzymes were promoted by the BR treatment, while the BRz inhibited the activity of the two enzymes. It was shown that BR regulated the production of endogenous NO by inducing the increase activity of NOS-like and NR enzyme during the adventitious root formation.
NO content, NOS-like and NR activity under BR, SNAP, L-NAME, Tungstate and BRZ treatments
In order to further verify whether NO participates in BR-induced adventitious roots formation in cucumber, the explants were placed in BR, SNAP, BR + L-NAME, BR + Tungstate and BRz treatment for 24 h, and the fluorescence localization of NO in hypocotyl, the content of endogenous NO and the activities of NR and NOS-like enzymes were analyzed. As shown in Figs. 4A and 4B, after cucumber explants treated with BR and SNAP, brighter green fluorescence was observed in the tissue position where hypocotyl produce adventitious roots, and the intensity of green fluorescence were significantly higher than that treated with the control, indicating that the production of NO was sharply rising. Compared with the control, the BR and SNAP treatments showed obvious green fluorescence distribution. However, the BR+L-NAME, BR+ Tungstate and BRz treatments showed the green fluorescence in the hypocotyl was evidently lower than that in the control plants (Fig. 4A and 4B). Subsequently, the quantitative analysis of NO content was done in hypocotyl of cucumber explants. As shown in Figs. 4C, compared with the control, endogenous NO content after treatment with BR and SNAP was significantly increased by 78.03% and 84.79%, respectively. Compared with the BR treatment, when L-NAME and Tungstate were added to the BR solution, the effects of BR were reversed. The NO content was reduced by 66.5% and 63.8%, respectively (Fig. 4C). Moreover, BRZ treatment alone significantly reduced NO content by 68.1% compared with the BR treatment (Fig. 4C). The qualitative and quantitative analysis of NO in hypocotyl of cucumber explants showed that exogenous application of BR and SNAP significantly increased the production and distribution of endogenous NO in cucumber hypocotyl. As shown in Figs. 4D and 4E, BR-induced NOS-like and NR activity were blocked by L-NAME and Tungstate. Compared with the control, application of BR and SNAP alone significantly increased the activity of NOS-like enzyme by 40.24% and 45.22%, respectively (Fig. 4D). Moreover, BR+L-NAME and BRz treatments markedly reduced NOS-like enzyme activity by 65.92% and 66.97% compared with the BR treatment, respectively (Fig.4D). Similarly, compared with the control, the activity of NR after treated with BR and SNAP was significantly increased by 40.17% and 41.53%, respectively (Fig. 4E). Compared with the BR treatment, the activity of NR enzyme after BR + Tungstate and BRZ treatment was drastically reduced by 41.65% and 43.59%, respectively (Fig. 4E). Thus, BR induced the generation of NO by regulating the activity of NOS-like and NR enzymes, and promoted adventitious root formation in cucumber explants.
The relative expression of NR gene under BR, BRZ, SNAP, and Tungstate treatments
During the adventitious rooting process, we performed real time RT-PCR to measure the relative expression of NR gene (Fig. 5). Compared with the control, the NR expression levels in BR- and SNAP- treatment were significant higher than those in the control at 24 h after treatment, which were 642.3% and 701.2% higher over the control (Fig. 5). There was no significant difference in the relative expression level of NR between BR + tungstate and the control. The relative expression level of NR gene decreased by 89.15% and 89.69% in BR + Tungstate and BRZ treated explants compared with the BR treatment, respectively (Fig. 5). These above results confirmed that BR up-regulated the relative expression level of NR, thus increasing the activity of NR enzyme, causing the increase of endogenous NO generation, promoting the development of adventitious roots.