BR concentrations affect number and length of adventitious roots
In order to investigate the effect of BR on adventitious roots, the cucumber explants were exposed to different concentrations of BR (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, both reaching the maximum values at 1μM (Fig. 1). Thus, the optimum concentration of BR (1 μM) was used in the subsequent experiment.
Number and length of adventitious root are 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-induced 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.
Temporal regulation of endogenous NO content, NOS-like and NR activity by BR
The time-course of NO content as affected by 1 μM BR or 1.5 μM BRZ treatment is shown in Fig. 3A. Compared with the control, the NO content of BR-treated explants has a slow downward trend at 0 h-6 h, which may be due to the wound response. From 6 h to 24 h, the NO content of BR-treated explants increased, and subsequently gradually decreased 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 0 h to 48 h, and the NO levels was always lower than those of BR-treated explant. Thus, the data suggest that BR regulated endogenous NO to promote the development of adventitious roots in cucumber.
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 the formation of adventitious roots (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 from 6 h to 24 h, reaching a maximum at 24 h, which was about 2-fold of the control. Then, from 24 h to 48 h, NOS-like activity tended to gradually decreased (Fig. 3B). Meanwhile, compared with the control and the BR treatment, the NOS-like enzyme activity of BRZ treatment consistently decreased throughout the whole experiment (Fig. 3B). Similarly, the NR activity of explants treated with BR decreased transiently during the first 6 h period, followed by a significant increase from 6 h 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 in BRz-treated explants continuously decreased during the time of experiment (Fig.3C). In conclusion, the activity of NOS-like and NR enzymes were promoted by BR treatment, while the BRz inhibited the activities of these two enzymes. Here, we showed 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. 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 treatment with BR and SNAP, brighter green fluorescence was observed in the tissue at the place where hypocotyl produce adventitious roots, and the intensity of fluorescence was significantly higher than in control explants, indicating that the production of NO was sharply rising. In opposite, explants treated with BR+ L-NAME, BR+ Tungstate and BRz showed a lower fluorescence in the hypocotyl than in the control plants (Fig. 4A and 4B). In order to support the qualitative analysis, the quantification of endogenous 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. Indeed, 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 analyses 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 BR and SNAP treatment 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 significantly higher than those in the control at 24 h after treatment, which were 642.3% and 701.2% higher over in 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).