FC诱导蚕豆气孔开放与保卫细胞H₂O₂的关系研究

【作者】 韩玺柱；

【导师】 佘小平；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2008， 硕士

【摘要】 先前的研究证明，壳梭孢素（fusicoccin，FC）有促进蒸腾作用、种子萌发、细胞伸长、胚芽鞘发育、叶形态发生和诱导细胞程序性死亡等效应。另有研究发现，暗和脱落酸（ABA）通过诱导保卫细胞H₂O₂产生促进气孔关闭，而FC通过活化质膜H⁺-ATPase诱导气孔开放。但时至今日尚未见FC促进气孔开放与保卫细胞H₂O₂变化关系的研究。本文以蚕豆为试验材料，结合运用表皮条试验方法和激光扫描共聚焦显微镜技术，研究了FC诱导蚕豆气孔开放与保卫细胞H₂O₂的关系，还对FC影响保卫细胞H₂O₂水平的方式进行了探索。所得结果主要如下：1．暗中分别用FC、H₂O₂清除剂ASA、H₂O₂产生酶NADPH氧化酶抑制剂DPI处理表皮条3 h，发现与ASA、DPI一样，FC显著促进气孔开放，暗示FC促进气孔开放的效应与保卫细胞H₂O₂水平降低有关。保卫细胞内源H₂O₂检测结果表明，FC与ASA、DPI一样确实显著降低了保卫细胞H₂O₂水平。2．光下分别用外源H₂O₂、FC、ASA、DPI、H₂O₂+FC、H₂O₂+ASA、H₂O₂+DPI处理表皮条3 h，以光处理为对照。发现外源H₂O₂极显著诱导气孔关闭，FC与ASA一样显著逆转外源H₂O₂诱导的气孔关闭，而DPI却无此效应，暗示FC逆转外源H₂O₂诱导的气孔关闭可能通过清除H₂O₂实现。保卫细胞内源H₂O₂检测结果表明，外源H₂O₂处理确实提高了保卫细胞H₂O₂水平，FC与ASA一样确实显著降低了外源H₂O₂处理保卫细胞的H₂O₂水平，而DPI却无此效应，可见FC确有清除H₂O₂的作用。3．先以暗处理表皮条3 h，再分别以FC、ASA、DPI处理表皮条另外3 h，并设置光、暗处理表皮条3 h两个对照。发现暗处理3 h极显著诱导气孔关闭，FC与ASA一样极显著促进已关闭气孔重新开放，而DPI却无此效应，暗示FC促进已关闭气孔重新开放可能通过清除H₂O₂实现。保卫细胞内源H₂O₂检测结果表明，FC与ASA一样确实显著降低了暗处理3 h诱导的保卫细胞内源H₂O₂，而DPI却无此效应，可见FC确有清除H₂O₂的作用。如将暗中FC促进气孔开放、降低保卫细胞H₂O₂的结果与FC逆转外源H₂O₂诱导气孔关闭、降低外源H₂O₂处理保卫细胞H₂O₂和促进暗诱导已关闭气孔重新开放、降低暗处理3 h后保卫细胞H₂O₂的结果联系起来考虑，可以得出暗中FC通过清除作用降低H₂O₂进而促进气孔开放的结论。更多还原

【Abstract】 Previous studies have shown that fusicuccin（FC） can promote transpiration, seed germination, cell elongation, the development of coleoptile and leaf generation. FC can also induce cell program death. Moreover, studies shows that darkness- and ABA-inducd stomatal closure were mediated by the increase of H₂O₂ in guard cell, FC promote stomatal open by activating plasma membrane H⁺-ATPase. However, the relationship between FC-induced stomatal open and the change of H₂O₂ level in guard cell remains unclear. In the present study, the relationship between the change of H₂O₂ levels in guard cells of Vicia faba and FC-induced stomatal open and the action mechanism of FC were investigated by means of laser scanning confocal microscopy technique and the epidermal strip bioassay.The main results are as followed:1. Epidermal strips were treated with FC, ASA （an important reducing substrate for H₂O₂ removal）, DPI（an inhibitor of the H₂O₂-generating enzyme NADPH oxidase） in darkness for 3h, we found that like ASA and DPI, FC induce stomatal open, indicate that FC-induced stomatal open is related to the decrease of H₂O₂ level in guard cell, we used H2DCF-DA, a specific probe for intracellular H₂O₂, to measure H₂O₂ levels directly in guard cells to determine whether or not FC-induced stomatal open is accompanied by a decrease of H₂O₂ levels in darkness. Our results provide evidence that like ASA and DPI, FC reduce H₂O₂ levels in darkness.2. Epidermal strips were treated with exogenous H₂O₂, FC, ASA, DPI, H₂O₂+ FC, H₂O₂+ ASA, H₂O₂+ DPI in light for 3h, we found that exogenous H₂O₂ remarkably induce stomatal closure. Like ASA, FC can reverse exogenous H₂O₂-induced stomatal closure, but DPI couldn’t. The results suggest that FC can abolished H₂O₂ in guard cell we used H₂DCF-DA, a specific probe for intracellular H₂O₂, to further clarify whether FC can affect exogenous H₂O₂-induced DCF fluorescence. Our results showed that FC can scavenge H₂O₂.3. Epidermal strips were treated in darkness alone for 3h, then these epidermal strips were treated with FC, ASA, DPI in darkness for another 1, 3h, we found that darkness remarkably induced the stomatal closure. Like ASA, FC can reopen the stomata that had been closed in darkness, but DPI couldn’t. The results suggest that FC can abolished H₂O₂ in guard cell, so reopened the stomata that had been closed in darkness. The effects of FC on the levels of H₂O₂ generated in guard cells held in the dark were also measured. The results showed that FC can scavenge H₂O₂.Taken together our results indicate that FC probably reduce the levels of H₂O₂ in guard cells by scavenging, inducing stomatal open in darkness.更多还原