| Based on the phenolic acids and IAA under the catalytic of PPO,the reaction system would be further improved and the reaction products were separated and its structure was also identified in this study.Meanwhile,the aspects of stability and physical activity of reaction products and its effect on the rooting of Paeonia plantlets in vitro were studied to reveal the relationship of phenolic metabolism with rooting in the root initiation process of Paeonia suffruticosa plantlets in vitro.The main results were as follows:(1)Used the detected endogenous phenolic compounds of peony planets in vitro(chlorogenic acid,caffeic acid and catechol)and IAA as raw material,researched the reaction between endogenous phenolic compounds of peony planets in vitro and IAA under the catalysis of PPO,the results showed that three phenolic acids(chlorogenic acid,caffeic acid and catechol)all reacted with IAA,and produced new substances.The HPLC chart showed that it formed three new substances at 5.572 min and 8.035 min and 9.045 min after 24 h.After separation and purification of three products,the HPLC chart showed that product I and product II have the same ultraviolet absorption figures(216 nm,279 nm)which were existed at the same time,and mutual transformation.This phenomenon showed that the product I and product II were isomers.The ultraviolet absorption peaks of product III in 242,260 and 298 nm.The mass-to-charge ratio [M+H]+ m/z of product I:169.0497,and the infrared spectrum showed that the product I have benzene.The structure of product I was 4,5-dimethoxy-1,2-benzoquinone.The mass-to-charge ratio [M+H]+m/z of product III: 146.1128,and melting point between 195-198 ?.Though the NMR and Py-GC-MS,we considered that the structure of product III is indole-3-formaldehyde.(2)We compared with the physiological activity of the products and IAA,the results showed that there was no significant difference between 24 h and 48 h at different concentrations of the product I and control of the elongation of wheat maltsheath.The effect of product I on wheat coleoptile elongation was not obvious.The different concentrations of product I treatments have no significant difference of the rooting in mung bean hypocotyl,which were overall higher than control.The maximum rooting rate still appeared in the treatment of IAA.The results showed that the product I can promote the rooting of mung bean cuttings,but lower than IAA.After 48 h,the treatments of product III(10-1 and 10-2 mmol·L-1)were significant differences with control,but overall was lower than that of IAA.The results showed that the effect of product III on wheat coleoptile elongation was significant,but lower than IAA.In the aspect of mung bean hypocotyl adventitious rooting,rooting rate was reached the highest under 10-2 and 10-3 mmol·L-1 treatment of product III,which were no significant differences with IAA 10-4mmol·L-1treatment.The results showed that the effect of product III on rooting of mung bean cuttings was higher than that of IAA,but there was no significant difference between them.To sum up,The ability to promote elongation: IAA > product III > product I.The ability to promote rooting:product III>IAA>product I.(3)The products of different concentrations were added to the rooting medium.With the increase of the concentration of product I the rooting rate of plantlets in vitro increased first and then decreased.And at the concentration of 3 mg·L-1 reached the maximum value of 26.92 %,which was significantly lower than the treatment of IAA3 mg·L-1.Also,with the increased of the concentration of product III the rooting rate increased gradually.When the concentration was 4 mg·L-1 the rooting rate reached the highest which was higher than that of IAA.Different treatments(product I,product III,IAA and CK)were used to investigate the changes of enzyme activity during rooting,the results showed that there was a great difference among different treatments.The changes of POD activity in 0-5 d were consistent in all treatments and in the root primordium induction period(3-5 d)fluctuated significantly.The product III and IAA treatments both reached the peak at the activity of POD at the third day and the activity of product III treatment was higher than that of IAA.There was no significant difference in PPO activity among the treatments.The treatments of IAA and product III both reached the peak at the third day while the treatment of product III was higher than that of IAA.At the same time the activity was the lowest in the control treatment.The activity of IAAO fluctuated in a small range.During the induction period(3-5 d)of root primordium the activity in IAA,product1 and 3 treatments was in a downward trend wholly,and activity of product III treatment was the lowest.(4)A method of six endogenous hormones(ZT,IAA,GA3,ABA,SA,IBA)in plants was established by UHPLC-Q-TOF-MS.The results showed that the retention time of six endogenous hormones: ZT:0.51 min?GA3: 2.12 min?IAA:2.71 min?ABA:3.03 min?IBA:3.70 min and SA:4.74 min.The six endogenous hormones were detected and well separated in 5 minutes.Based on the study of different concentrations of formic acid(FA)in the mobile phase(0 ‰?0.1 ‰?0.2 ‰?0.5 ‰?1 ‰),we found that the response of ZT was lower with increased concentration of FA.The highest response of GA3?IAA?ABA and IBA were appeared at 0.1 ‰FA.Under 0.5 ‰FA,the response of SA was reached the maximum.When the mobile phase was free of formic acid,the other five hormones were the lowest in response except ZT.Therefore,0.1 ‰ FA was suitable for the detection of six endogenous hormones.Experiments of the change of endogenous hormone levels during rooting period found that IAA of stem base showed rising trend during the root primordium induction period.The level of ZT was steadily low during 0-10 d.During the root primordium induction period,the GA3 showed a trend of "up-down ",and then it kept a decline trend.The ABA always kept at a low levels,it kept a decline trend during 0-5 d and rising trend during 5-25 d.The location of IAA was observed on vascular bundle and always distributed in the root primordium induction area by immunofluorescence.The strength of the signal was match with the IAA level.(5)The key endogenous metabolites of tree peony during rooting were extracted.The results showed that 4752 peaks were found in positive ion mode,and 4740 peaks were found in negative ion mode.The metabolites of tree peony in root primordium periods were mainly distributed between 0.5-2 min,4-7 min and 10-14 min.Through principal component analysis(PCA),it was found that the fluctuation of endogenous metabolite in tree peony was not obvious during the third day of rooting.In contrary,it was fluctuated greatly in the fifth day,and the days of culture and hormone treatments were distinguished.We concluded that the endogenous metabolite changes of tree peony in vitro were closely related to the rooting during the formation of the root primordium(3-5 d).In conclusion,three phenolic acids(chlorogenic acid,caffeic acid and catechol)all reacted with IAA in the catalysis of PPO,and produced the same new substances.The results showed that the product I might be 4,5-dimethoxy-1,2-benzoquinone,which was an isomer with product II.The product I and product II were transformed into each other.The product III was indole-3-carboxaldehyde.The auxin activity ofproduct I was not obvious.The product I had a certain effect on the occurrence of adventitious roots,but it was no significant difference with IAA.The product III had significant auxin activity,but the activity was lower than IAA.The promoting effect of adventitious root was higher than that of IAA.The product I added at suitable concentration had a certain promoting effect on rooting of tree peony in vitro,but the rooting rate was significantly lower than that of IAA treatment.This result speculated that the 4,5-dimethoxy-1,2-benzoquinone might be an auxiliary factor of rooting.The product III had a significantly promot the rooting of tree peony in vitro,and the rooting rate was higher than that of IAA,but there was no significant difference between the two treatments.It was speculated that the diphenol reacted with IAA under the action of PPO,and part of IAA changed into indole-3-carboxaldehyde.The indole-3-carboxaldehyde had auxin activity,which could promote the rooting of tree peony in vitro and could not be oxidized by IAAO.Thus,it had a protective effect on the content of IAA during root primordium induction period.The level of endogenous auxin in tree peony was maintained,which promoted the induction of root primordium. |
PDF Full Text Request