The role and signaling of sphingosine-l-phosphate （SIP） during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S 1P and hydrogen peroxide （H202） levels and closed stoma...The role and signaling of sphingosine-l-phosphate （SIP） during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S 1P and hydrogen peroxide （H202） levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine （DL-threo-DHS） and N,N-dimethylsphingosine （DMS）, two inhibi- tors of long-chain base kinases. Exogenous SIP led to stomatal closure and H202 production, and the effects of SIP were largely prevented by the H202 modulators ascorbic acid, catalase, and diphenyleneiodonium. These results indicated that SIP mediated darkness-induced stomatal closure by triggering H202 production. In addition, DL-threo-DHS and DMS significantly suppressed both darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous SIP caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that SIP syn-thesis was necessary for cytosolic alkalization during stomatal closure caused by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H202 production by butyric acid revealed that S 1P synthesis-induced cytosolic alkalization was a prerequisite for H202 production during stomatal closure caused by darkness, a conclusion supported by the facts that the pH increase caused by exogenous SIP had a shorter lag and peaked faster than H202 levels and that butyric acid prevented exogenous SIP-induced H202 production. Altogether, our data suggested that darkness induced SIP synthesis, causing cytosolic alkalization and subsequent H202 production, finally leading to stomatal closure.展开更多
The present study aimed to investigate the targeting effect of H7K（R2）2-modified pH -sensitive liposomes on U87-MG cells. Using coumarin-6 as a fluorescence probe, we prepared H7K（R2）2-modified p H-sensitive lipos...The present study aimed to investigate the targeting effect of H7K（R2）2-modified pH -sensitive liposomes on U87-MG cells. Using coumarin-6 as a fluorescence probe, we prepared H7K（R2）2-modified p H-sensitive liposomes（designated as coumarin-6-PSL-H7K（R2）2）. The flow cytometry assay was used to evaluate the effect of H7K（R2）2 proportions on the cellular uptake and endocytosis pathways of coumarin--6--PSL--H7K（R2）2 on U87-MG cells. The circular dichroism（CD） spectroscopy assay was used to investigate the secondary structures of H7K（R2）2 peptide at pH 7.4 and H 6.8, respectively. Our results indicated that the 2.5% proportion of H7K（R2）2 in the coumarin-6--PSL-H7K（R2）2 was superior to those of 1% and 3.5% of H7K（R2）2. The uptake of coumarin--6-PSL--H7K（R2）2 on U87--MG cells was not inhibited by filipin, M-β--CD or chlorpromazine. The secondary structure of H7K（R2）2 at pH 6.8 was mostly presented as β--turn. In conclusion, we suggested that the appropriate proportion of H7K（R2）2 in the H7K（R2）2--modified pH--sensitive liposomes could be set at 2.5%. The cellular uptake pathway for H7K（R2）2-modified pH--sensitive liposomes was via the cell penetrating capacity of H7K（R2）2 which responded to acidic condition. The secondary structure of H7K（R2）2 at pH 6.8, which was presented as the shape of hairpin, might be mainly responsible for its targeting and cell penetrating effect.展开更多
基金supported by the Graduate Education Innovation Program Projects of Ministry of Education, China (Grant No. F-0922)
文摘The role and signaling of sphingosine-l-phosphate （SIP） during darkness-induced stomatal closure were examined in Vicia faba. Darkness substantially raised S 1P and hydrogen peroxide （H202） levels and closed stomata. These darkness effects were significantly suppressed by DL-threo-dihydrosphingosine （DL-threo-DHS） and N,N-dimethylsphingosine （DMS）, two inhibi- tors of long-chain base kinases. Exogenous SIP led to stomatal closure and H202 production, and the effects of SIP were largely prevented by the H202 modulators ascorbic acid, catalase, and diphenyleneiodonium. These results indicated that SIP mediated darkness-induced stomatal closure by triggering H202 production. In addition, DL-threo-DHS and DMS significantly suppressed both darkness-induced cytosolic alkalization in guard cells and stomatal closure. Exogenous SIP caused cytosolic alkalization and stomatal closure, which could be largely abolished by butyric acid. These results demonstrated that SIP syn-thesis was necessary for cytosolic alkalization during stomatal closure caused by darkness. Furthermore, together with the data described above, inhibition of darkness-induced H202 production by butyric acid revealed that S 1P synthesis-induced cytosolic alkalization was a prerequisite for H202 production during stomatal closure caused by darkness, a conclusion supported by the facts that the pH increase caused by exogenous SIP had a shorter lag and peaked faster than H202 levels and that butyric acid prevented exogenous SIP-induced H202 production. Altogether, our data suggested that darkness induced SIP synthesis, causing cytosolic alkalization and subsequent H202 production, finally leading to stomatal closure.
基金Specialized Research Fund for the Doctoral Program of Higher Education(Grant No.20120001110012)the National Basic Research Program of China(973 Program,Grant No.2013CB932501)National Natural Science Foundation of China(Grant No.81172992)
文摘The present study aimed to investigate the targeting effect of H7K（R2）2-modified pH -sensitive liposomes on U87-MG cells. Using coumarin-6 as a fluorescence probe, we prepared H7K（R2）2-modified p H-sensitive liposomes（designated as coumarin-6-PSL-H7K（R2）2）. The flow cytometry assay was used to evaluate the effect of H7K（R2）2 proportions on the cellular uptake and endocytosis pathways of coumarin--6--PSL--H7K（R2）2 on U87-MG cells. The circular dichroism（CD） spectroscopy assay was used to investigate the secondary structures of H7K（R2）2 peptide at pH 7.4 and H 6.8, respectively. Our results indicated that the 2.5% proportion of H7K（R2）2 in the coumarin-6--PSL-H7K（R2）2 was superior to those of 1% and 3.5% of H7K（R2）2. The uptake of coumarin--6-PSL--H7K（R2）2 on U87--MG cells was not inhibited by filipin, M-β--CD or chlorpromazine. The secondary structure of H7K（R2）2 at pH 6.8 was mostly presented as β--turn. In conclusion, we suggested that the appropriate proportion of H7K（R2）2 in the H7K（R2）2--modified pH--sensitive liposomes could be set at 2.5%. The cellular uptake pathway for H7K（R2）2-modified pH--sensitive liposomes was via the cell penetrating capacity of H7K（R2）2 which responded to acidic condition. The secondary structure of H7K（R2）2 at pH 6.8, which was presented as the shape of hairpin, might be mainly responsible for its targeting and cell penetrating effect.
