The Identification Of The Transport Function Of H₂O₂ Transported By Hb Pips Of Rubber Tree And Its Effect On The Latex ROS Level

The laticifer cell of Hevea brasiliensis is produced by the differentiation of lamina cells distributed in the phloem,and plays an important role in the basic research of the physiology of rubber production and drainage.The process of rubber production and debinding is closely related to the water balance of the bast of the rubber tree,and the plant aquaporin（AQP）,as a specific and efficient bidirectional transport channel of water across the plasma membrane and vacuole membrane,can sensitively and efficiently regulate the intercellular and the intracellular water flows and participates in the regulation of active oxygen in the body,which is beneficial to the normal growth and metabolism of plants.Therefore,specific and highly efficient aquaporins that are widely present on cell membranes may be involved in the transport of reactive oxygen species in rubber latex.However,related studies have only focused on the effluent aquaporin genes cloned in rubber trees,and are limited to uncut trees and parts and identification of aquaporins for their water transport capacity.In view of this,in this study,the Brazilian rubber tree（Hevea brasiliensis）“Reyan 7-33-97”,which is currently being widely planted in China’s rubber plantation area,was used as the material.The ethylene resin was stimulated with analyze the rubber H₂O₂ content and CAT.Studies on the enzyme activities such as POD and the determination of H₂O₂ content and CAT,POD and other enzyme activity contents during the debinding process;Using AR and H₂DCFDA probe labeling methods,under the condition that H₂O₂in vitro,the fluorescence intensity of4 aquaporin gene recombinant yeast cells H₂O₂ and ROS were quantitatively analyzed using fluorescence electron microscopy and fluorescence,respectively,to identify the latex expressing aquaporin HbPIP1;4,HbPIP2;3,HbPIP2;5,HbPIP2;7 transport H₂O₂ function;in order to reveal the ability of the aforementioned aquaporin membrane aquaporins HbPIPs to transport H₂O₂and its effect on latex ROS levels.The findings are as follows:1.Ethephon stimulates rubber trees,and the latex H₂O₂ content changes significantly during the debinding process.At high concentration（10%）of Ethephon stimulation,there was no significant difference in H₂O₂ content in the latex compared with the control group at the early stage of debinding.The H₂O₂ content remained at a high level,and the same phenomenon appeared in the H₂O₂ content in the bark.With the prolongation of the debinding time,the content of H₂O₂ in the latex reached the maximum at 90 minutes of debinding;at the end of the debinding period,the H₂O₂ content of the latex was significantly higher than that at the beginning of the debinding,while the H₂O₂ content of the bark was significantly lower than the beginning of the debinding.It is preliminary indicated that high concentration of ethephon stimulation can cause a large amount of H₂O₂ to accumulate in the latex,but in the early stage of debinding,the H₂O₂ produced by the latex and bark may be completely removed by the active oxygen scavenging mechanism in the body.In addition,the normal debinding in the gum tree is beneficial to reduce the oxidative damage caused by H₂O₂ to the bark.For the“Reyan 7-33-97”rubber tree,the oxidative damage of the bark tissue can also reflect the oxidative damage of the milk duct cells.2.Ethylene stimulates rubber trees,and the latex protein carbonyl content and MDA content have significant changes during the debinding process.At the beginning of debinding,the latex protein carbonyl content maintained a relatively low level,but with the extension of the debinding time,the latex protein carbonyl content gradually increased and then decreased,and the content at the end of the debinding period was significantly higher than the beginning of debinding;Latex protein carbonyl content reached the highest level in advance compared with the control group.The latex MDA content showed a decreasing-increasing-decreasing trend during the debinding process,but the control group only significantly increased the MDA content at the end of debinding.It indicated that high concentration of ethephon stimulated rubber trees,and the latex protein carbonyl content and malondialdehyde content increased.It showed that high concentration of ethephon stimulates the gum tree,which will further enhance the degree of oxidative damage of milk duct cells.3.This study found that when ethephon was applied to stimulate rubber trees,the activities of latex CAT,POD,T-AOC,and PPO increased significantly.With the extension of the debinding time,the antioxidant enzyme activity is different,suggested that CAT,POD,T-AOC,and PPO play a large role in the regulation of latex active oxygen.With the extension of the debinding time,the application of 5%ethephon stimulated the rubber tree.The latex CAT enzyme activity reached its maximum at 60 minutes,the latex POD was low,and the T-AOC and protein carbonyl content were maintained at a relatively stable level;At the end of the gel,ethephon stimulation increased the CAT,POD activity and protein carbonyl content,and the total antioxidant capacity was indeed significantly lower;PPO activity was the strongest at the beginning of the debinding,and when the debinding was stopped,the activity was significantly lower It is speculated that PPO may participate in the regulation of ethylene and promote debinding.4.Analyze recombinant yeast cells with fluorescent probes.AR probe,HbPIP1;4,HbPIP2;3,HbPIP2;5 expression,the intracellular fluorescence signal intensity gradually increased within 45 minutes,while HbPIP2;7 expression,intracellular H₂O₂ quantity fluctuated greatly during the first 15 minutes,followed by green fluorescence H₂DCFDA detection The same phenomenon occurs with the needle.It showed that HbPIP1;4,HbPIP2;3,HbPIP2;5,HbPIP2;7 proteins all have the ability to transport H₂O₂,but HbPIP2;7 protein transports H₂O₂ is weak.In summary,the high concentration of ethephon stimulates the rubber tree,and the latex H₂O₂ activity is enhanced,which will cause great oxidative damage to the rubber tree.Rubber tree milk tube plasma membrane aquaporin HbPIP1;4,HbPIP2;3,HbPIP2;5,HbPIP2;7 all have the ability to transport H₂O₂.This study has important theoretical significance for the research of rubber tree production and debinding.