Involvement Of Ros And Ca²⁺in ABA And SA Triggered Adventitious Root Formation In Mung Bean Hypocotyls

Adventitious root (AR) is an important part of plant root system. Adventitious root formation (ARF) is often used as a fundamental model to study the events of plant growth and development. Salicylic acid (SA) and abscisic acid (ABA) have been regarded as pivotal signal molecules that regulate many metabolic processes in plants, especially in regulating defense system under biotic and abiotic stresses. Recent research indicated that ABA and SA could induce ROS accumulation and increase intracellular Ca2+ concentration under certain stress stimulus. While the definitive role of ABA and SA in ARF; and their action modes are still waiting for elucidation. By using mung bean (Phaseolus radiatus L) hypocotyl cuttings as materials, we investigated the impacts of ABA and SA on ARF, and the involvement of ROS and Ca2+ in ABA and SA signaling pathways.We found that ABA enhanced synergistically indole-3-acetic acid (IBA) induced ARF in mung bean hypocotyl cuttings Furthermore, ABA probably acted as an upstream regulator or acted as a cofactor of IB A in induction of ARF. Previous data have proved that H2O2 signaling happens in adventitious rooting. We found that pretreatment of the cuttings with DMTU or DPI was able to inhibit ABA action on ARF, which suggested that ROS should play a crucial role in ABA induced ARF. Biochemistry determination showed that ABA incubation could result in H2O2 and O2- accumulation in the cutting sections, not only via the increase of NADPH-oxidase activity, but also SOD activity.Pharmacological methods revealed that SA was directly involved in the ARF in a dose-dependent manner. SA was able to induce re-establish new apical meristems and enlarge radicate zones near the cutting. Besides, MeJA showed an antagonistic effect on SA induced ARF.Low dosages of H2O2 improved SA induced ARF, but at higher concentrations H2O2 caused decreases of SA induced AR numbers perhaps due to the happens of oxidation damage. The promotive effect of SA on ARF was inhibited by DMTU or DPI, results suggested that ROS should be a critical part in SA pathways in ARF. SA treatment resulted in an increase of H2O2 generation in the hypocotyl cuttings in the tests using histochemistry methods with 3.3-diamino benzidine (DAB) staining and cytochemical detection. Further, S A pretreatment caused an increase of SOD activity, and a decrease of CAT activity. Above data indicated that the H2O2 accumulation caused by SA pretreatment should attribute mainly to mediate the antioxidant defense.However, there might exist different signaling pathways between ABA and SA in regulation of ARF. ABA raised NADPH-oxidase activity while SA treatment showed little influence on NADPH-oxidase activity. Again, SA suppressed CAT activity but ABA raised it. It is suggested that ABA and SA triggered ROS accumulation in alternatively, and then evoked ARF in the seedling cuttings.Ca2+ acts as a second message in many processes related to the regulation of plant growth and development. Exogenous application of Ca2+ did not generate more adventitious roots, while Ca2+ at high concentrations inhibited ARF perhaps due to a stress of a high ion concentration. Extracellular Ca2+ chelator EGTA showed little action on ARF, as well as on IB A, ABA, SA and ROS induced ARF in the cuttings. Whereas, intracellular Ca2+ chelator BAPTA blocked significantly the IBA, ABA, SA and ROS induced ARF. Above data revealed that the elevation of intracellular Ca2+ concentration dominates ARF. LiCl, a well described vacuole membrane Ca2+ channel inhibitor, inhibited tremendously ARF, suggesting that ARF induced by induced by IBA, ABA, SA or ROS should be mediated via regulation of Ca2+ channels located on vacuole membrane.Taking together, ABA or SA triggered certain antioxidant enzymes, leading to the accumulation of ROS. ROS then activated the vacuole membrane Ca2+ channels, causing an elevations in [Ca]cyt via the release of Ca2+ from intracellular stores such as the vacuole. Above events resulted in a final induction of adventitious root formation in mung bean hypocotyl cuttings.