A Study On The Role Of Nitric Oxide In The Lateral Root Formation Of Mung Bean

Nitric oxide(NO) is one of the most important signaling molecules of plant. It was proved to participate in many key plant physiological process such as growth, development, pathogen defense reaction, programmed cell death, and stress tolerance. Though NO mediation of the lateral rooting process induced by auxins had been demonstrated, the time and space changes of NO and the source of NO accompanied lateral root generation and development are not very clearly. In this study, mung bean were used as materials, and the role and the source of NO of the radicles treated by H₂O, IBA or NAA during the lateral root formation were studied from several aspects. The results were as follows:1. The radicles were treated with NO-associated reagent. The results showed that NO donor-SNP significantly enhance the lateral rooting. 50μmol/L SNP proved an optimal concentration for the initiation of organogenesis. The treatment time of SNP also influenced the rooting. The radicles treated with 50μmol/L SNP for 12h had the optimal rooting effect. SNP obviously enhanced the effects of IBA or NAA in stimulating rooting. C-PTIO, NO specific scavenger, or NOS inhibitor L-NAME treated alone or plus IBA or NAA resulted in an inhibitory effect. It's indicated that endogenous NO appears to play a key role in the generation and development of lateral roots, and the production of NO in this process maybe catalyzed by NOS.2.We used the NO-sensitive fluorescence probe DAF-2DA to detect the NO of radicles treated by H₂O. IBA or NAA during the generation and the development of lateral roots of mung bean about its temporal and spatial change. The results indicated that, in the group treated by H₂O, the development of root primordium, the fluorescence of NO appeared and increased gradually in 2mm-4mm region of radicle from hypocotyl basal part. The green fluorescence of NO was detected in pericycle and endoderm cells at 24h after the seeds imbibed in water for 1 day and a half. After 36h, With thedevelopment and elongation of root primordium, the number of DAF-2DA-positive cells increased gradually, and the root apical meristem cells show intense fluorescence. No NO was observed in non-rooting areas. In the group treated by IBA or NAA, fluorescence was detected earlier than control group. In 4mm-7mm region of radicle from hypocotyl basal part, the generation and distribution of NO of the plants treated by IBA or NAA were different with that of treated by H2O. No NO was detected in control. The generation, distribution and change of NO of plants treated by IBA or NAA in this region were in accordance with that of in 2mm-4mm region of radicle from hypocotyl basal part. C-PTIO, the specific NO scavenger, suppressed the fluorescence and inhibited the formation of root primordial clearly. The results strongly argue that that endogenous NO appears to play a key role in the lateral rooting process. It also suggested that NO could also mediate the IBA and NAA response during the rooting process in mung bean.3. NOS-like enzyme activity was detected during lateral root formation by NADPH-diaphorase histochemical method. With the origination and development of lateral roots, not in control but in the plants treated by IBA or NAA, the activity of NADPH-diaphorase and the number of cell expressing NADPH-diaphorase-positive increased rapidly in the forming root in 2mm-4mm region of radicle from hypocotyl basal part. The non-rooting areas haven't positive cells. In accordance with the distribution of NO, intense staining mainly distributed in root meristem. The NADPH-diaphorase activity of the plants treated by IBA or NAA were observed earlier and play more intense than the control in the same time. In 4mm-7mm region of radicle from hypocotyl basal part, the distribution of NADPH-diaphorase of the plants treated by IBA or NAA were also different with that of treated by H2O. Very low level of NADPH-diaphorase was observed in control. The distribution and change of NADPH-diaphorase of plants treated by IBA or NAA in this region were in accordance with that of in 2mm-4mm region of radicle from hypocotyl basal part. L-NAME treatment significantly decreased the fluorescence of NO and NADPH-diaphorase staining, and the formation of root primordia were inhibited. The results presented above indicate the presence of a putative NOS and the enzyme is responsible for theproduction of NO during lateral root generation and development in mung bean.