| Microcystins?MCs?can enter farmland ecosystems by irrigation water contaminated with MCs,and adversely affect plant growth,yield and quality.It is necessary to clarify the mechanism of differences in plant tolerance to MCs for provding information to reduce adverse effects of MCs on plants.Antioxidative enzymes and protein phosphatases are important enzymes to maintain the balance of reactive oxygen species?ROS?and phosphorylation,and play an important role in plant tolerance to MCs stress.Here,we compared effects of MCs?concentration and exposure time?on growth and resistance ability in rice and cucumber seedlings under hydroponic cultivation,and analyzed the response of antioxidative enzymes and protein phosphatase in rice and cucumber to MCs at levels of protein and gene by using polyacrylamide gel electrophoresis and Real-time PCR.All result could provide a theoretical basis for using molecular biology methods to improve plant tolerance to MCs.The main results are as follows:?1?The effect of MCs on the growth and stress resistance of rice and cucumber seedlings showed that low concentration MCs(5?g·L-1)increased the contents of superoxide anion?O2·-?and hydrogen peroxide?H2O2?in rice leaves,didn't change the malondialdehyde?MDA?content and light energy conversion?Fv/Fm?,and promoted growth of rice.While the increase of O2·-and H2O2 contents and oxidative damage in cucumber occurred earlier than rice,and decreased the cucumber RGR and Fv/Fm,indicating that the tolerance of rice to MCs was higher than that of cucumber,and the tolerance time of rice to MCs at the same concentration was longer than that of cucumber.Medium or high concentration MCs(10,50 and 100?g·L-1)rapidly increased the ROS content of rice and cucumber and caused oxidative damage,decreased Fv/Fm and RGR.Among them,changes of O2·-content,MDA content,Fv/Fm and RGR were higher than rice,which indicates that the resistance of rice to MCs was stronger than that of cucumber was related to less O2·-accumulation in rice.?2?The effects of MCs on activities and molecular mechanism of SOD,CAT and POD in rice and cucumber showed that low concentration MCs(5?g·L-1)up-regulated transcription levels of rCu/Zn-SOD 1,rCu/Zn-SOD 2,rCu/Zn-SOD 3 and rCAT 3 encoding SOD and CAT in rice,increased the amount and activity of isoenzymes,and then enhanced the ability of scavenging ROS to protect rice from oxidative damage induced by MCs.Although the transcription levels of SOD genes in cucumber up-regulated,SOD isoenzyme amount and activity didn't change,resulting to O2·-scavenging ability lower than that in rice.The increase in the transcription level of cCAT1 and the activity of CAT isozyme in cucumber increased less than that in rice,while the transcription levels of cPOD1 and cPOD2 and the POD isoenzyme activity in cucumber increased more than that in rice.Combined with the change trend of O2·-and H2O2 contents,it indicated that the increased SOD activity of rice under low concentration MCs stress enhances the ability of scavenging O2·-,which is one of the reasons why the degree of oxidative damage of rice is lower than that of cucumber.Medium or high concentration MCs(10,50 and 100?g·L-1)up-regulated transcription levels of SOD and CAT genes?rCu/Zn-SOD1,rCu/Zn-SOD 2,rCu/Zn-SOD 3,rFe-SOD,rMn-SOD,rCAT 1 and rCAT 3?in rice,increased the amount and activity of SOD and CAT isoenzymes,didn't change the POD transcription level and isoenzyme amount,so POD isoenzyme activity increased by H2O2 regulation.The same MCs treatment changed the transcription level of SOD genes in cucumber,but still did not change the total SOD activity,resulted in the ability of scavenging O2·-was still lower than that of rice.It indicated that up-regulation of the transcription levels of SOD encoding gene in rice enhanced the SOD isoenzyme amount and activity and enhanced the ability of scavenging O2·-is one of the reasons for rice has more resistant to MCs-induced oxidative damage than cucumber.?3?The effect of MCs on activities and molecular mechanism of protein phosphatase and serine-threonine protein phosphatase type 2A?PP2A?in rice and cucumber showed that the low concentration MCs did not change the total protein phosphatase activity and phosphorylation level of rice,up-regulated the transcription levels of PP2A genes,but decreased PP2A activity due to the ability of PP2A synthesis less than the consumption of MCs combined with PP2A.The same MCs decreased the activity of protein phosphatase in cucumber,inhibited the dephosphorylation process,and increased the level of phosphorylation,among then,up-regulated the transcription levels of PP2A genes,and decreased PP2A activity.Medium or high concentration MCs(10,50 and 100?g·L-1)decreased the total activity of rice and cucumber protein phosphatase,inhibited the dephosphorylation process,and increased the level of phosphorylation.Among them,the phosphorylation level in rice was lower than that in cucumber due to the decrease of protein phosphatase activity in rice was smaller than that in cucumber.The large up-regulation of catalytic subunit rPP2A-4c1 under MCs stress was conducive to synthesizing more PP2A catalytic subunits to reduce the consumption of covalent bonding of MCs and PP2A.While the PP2A regulatory subunit cPP2A-R-TAP46 has the highest up-regulation in cucumber,and the catalytic subunit cPP2A-c2 in cucumber was down-regulated at 100?g·L-1 MCs treatment,which resulted in a higher decrease in PP2A activity and inhibited the stomatal opening and growth.The results showed that the up-regulation of the transcription levels of PP2A catalytic subunit genes in rice increased PP2A synthesis,which might be one of the reasons why rice is more tolerant to MCs.In conclusion,rice has higher tolerance to MCs than cucumber.One reason was up-regulation of SOD and CAT isozyme genes in rice was higher than those in cucumber,the enzyme amount and activated isoenzyme activity to alleviate the oxidative damage caused by O2·-accumulation,thereby alleviating the adverse effects of MCs on rice photosynthesis and growth.Another reason was the up-regulation of PP2A subunit genes in rice was higher than those in cucumber,and more PP2A was synthesized to supplement the PP2A consumption caused by the covalent binding of MCs to PP2A,thereby slowing the degree of inhibition of protein dephosphorylation and alleviating the adverse effects of hyperphosphorylation on photosynthesis and growth in rice.All information can be helpful for finding effective countermeasures to mitigate harmful effects of MCs on crops. |
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