Changes Of CuAo Expression And Enzyme Activity During The Regeneration Process Of Soybean Hypocotyl Adventitious Root

The adventitious root growth of plants is affected by many factors, such as plant hormones, polyamines, NO, light, temperature and so on. Previous studies have indicated that polyamine oxidase plays a very important role in the process of adventitious root regeneration which may be involved in polyamine oxidative degradation, Therefore, a significant question is that in the process of adventitious roots, which relationship occurs between plant hormone, polyamines, illumination and other factors and polyamine oxidase gene expression and enzyme activity change. These reports are rare in China or abroad at present. To study the effects of these factors such as PAs, auxin and light on regeneration of soybean hypocotyl adventitious root, the "Zhong Huang 25" soybean were used and treated with exogenous PAs, AG, IBA, TIBA,AVG and different photoperiod. After that, we detected the number of adventitious roots and the changes of diamine oxidase gene expression and enzyme activity during the regeneration process of soybean hypocotyl adventitious roots. The results are as follows:The growth of soybean hypocotyl adventitious roots could be significantly promoted by low concentrations of Put 、 Spd and Spm, and that the optimum concentration was 1.0 mmol/L 、0.25 mmol/L and 0.05 mmol/L respectively. On the contrary, polyamines at high concentrations showed inhibitory effects on the growth of adventitious roots. The gene expression level of CuAO in soybean hypocotyl was detected. It was found that CuAO gene expression increased gradually from the second day, maintained a high level after the third day in the control, and the CuAO gene expression could be significantly up-regulated by three kinds of exogenous polyaminesduring the growth of adventitious root. Correspondingly, the CuAO activity was gradually increased during the regeneration process of adventitious roots, and was significantly higher in treatment group as compared with the control. Put had better effect than the other two kinds of polyamines. In addition, the role of aminoguanidine(AG, a specific inhibitor of CuAO) was detected. It could significantly inhibit the growth of soybean adventitious root, which might be related to the inhibition of CuAO activity by AG. These results imply that the expression of CuAO gene can be induced by exogenous polyamines. Polyamines can induce the expression of CuAO gene and promote the activity of the enzyme during the growth of adventitious root.The growth of soybean hypocotyl adventitious roots could be significantly promoted by indole butyric acid(IBA). When the concentration reached 30 mg/L, the number of adventitious root reached the highest value. But the growth of adventitious roots were inhibited when the concentration of IBA exceeds 30 mg/L. Both of auxin transport inhibitor(TIBA) and auxin synthesis inhibitor(AVG) significantly inhibited the growth of adventitious root of soybean. Detection of CuAO gene expression level showed that IBA treatment could significantly inhibit the expression of CuAO gene. But the level of CuAO gene expression increased gradually in the second day after IBA treatment and reached a similar level with the control at last. Correspondingly, the CuAO activity of the control group increased gradually with time prolonging. But the enzyme activity of the IBA treatment group decreased at first, and increased to a high level which was significantly higher than that of the control group in the third day after treatment. The detection of expression of CuAO gene showed that it could be promoted by TIBA in two days and promoted by AVG in one day after treatment. But the rest of the time, the level of CuAO gene expression was lower than the control group. TIBA and AVG have a strong inhibitory effect on the activity of CuAO in the late stage of adventitious root growth. These results indicate that CuAO might be involved in the soybean hypocotyl adventitious root regeneration which was promoted by auxin.Although auxin has a negative effect on the expression of CuAO gene, the pre-treatment of auxin has an obvious gain effect on CuAO gene expression and enzyme activity inthe late stage of adventitious root growth. And the effect of this promotion is synchronous with the adventitious root regeneration.The growth of adventitious root is better with the light exposure time prolonged in different photoperiods. Light treatment could significantly promote the expression of CuAO gene, the level of CuAO gene expression under dark conditions was significantly lower than that of light condition. The change of CuAO enzyme activity was consistent with the gene expression level. These results show that the expression of CuAO could be up-regulated by light. That the light promotes the growth of adventitious roots which may be related to the induction of CuAO gene expression and the activity of the enzyme.The results showed that polyamines, auxin and light can promote the regeneration of soybean hypocotyl adventitious root, what’s more, suitable polyamines and light can positively regulate CuAO gene expression levels and improve the activity of that enzyme. Although auxin has a negative effect on the expression of CuAO gene, the pre-treatment of auxin has an obvious gain effect on CuAO gene expression and enzyme activity in the late stage of adventitious root growth. Therefore, we think that exogenous polyamine, auxin and light can affect the regeneration of adventitious root of soybean by regulating the CuAO gene expression and enzyme activity.