一氧化氮参与调控油菜素内酯增强高山离子芥悬浮细胞抗寒性

Nitric Oxide Mediates Brassinosteroids-induced Chilling Tolerance in Chorispora bungeana Suspension Cultured Cells

摘要

为探究油菜素内酯（BRs）诱导提高植物的抗寒性是否受一氧化氮（NO）信号分子调控，以高山离子芥（Chorispora bungeana）悬浮细胞为材料，分别用24-表油菜素内酯（EBR）、NO供体SNP、NO清除剂PTIO、一氧化氮合成酶（NOS）抑制剂L-NAME、EBR+PTIO以及EBR+L-NAME进行处理，分析在低温胁迫下以上处理对细胞抗寒性、活性氧（ROS）水平以及抗氧化防御系统的影响。结果表明：（1）外源EBR处理可以缓解低温胁迫对悬浮细胞活力的抑制以及离子渗漏和膜脂过氧化程度的加剧，从而增强细胞对低温的抗逆能力。SNP处理对上述生理指标的影响与EBR类似。（2）在低温胁迫下，与EBR处理细胞相比，EBR+PTIO以及EBR+L-NAME处理会导致悬浮细胞活力下降，离子渗漏和膜脂过氧化程度显著升高，表明阻断NO信号会降低EBR诱导提高的抗寒性。（3）与仅受低温胁迫的细胞相比，外源EBR处理导致细胞NO含量和NOS活性进一步升高，而EBR诱导的NO积累可以被PTIO或L-NAME所抑制。（4）EBR、SNP处理均可以明显抑制低温胁迫下离子芥悬浮细胞过氧化氢（H₂O₂）含量、超氧阴离子（{\mathrm{O}}_{\mathrm{2}}^{·–}）产生速率和羟自由基（OH^-）含量的升高，并显著增强抗坏血酸过氧化物酶（APX）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）、过氧化物酶（POD）和超氧化物歧化酶（SOD）的活性以及增加抗坏血酸（AsA）和还原性谷胱甘肽（GSH）的含量，从而缓解低温造成的氧化损伤，而PTIO、L-NAME会不同程度抑制低温胁迫下EBR的这些保护功能。综上所述，EBR通过激活离子芥悬浮细胞的NOS活性来促进细胞内源NO积累，从而提高其抗寒性。EBR处理可以抑制低温胁迫下ROS过量积累并增强细胞的抗氧化防御能力，这 两个过程都受NO信号分子调控，从而缓解低温造成的氧化损伤，防止膜脂过氧化程度加剧，提高细胞对低温胁迫的抗逆性。因此，低温胁迫下，高山离子芥悬浮细胞NOS来源的NO是EBR信号转导的下游信号分子。

Abstract

In order to investigate whether brassinosteroids（BRs）-induced chilling tolerance is regulated through nitric oxide（NO） signaling molecule， the suspension cultured cells of Chorispora bungeana were treated with 24-epibrassinolide（EBR）， NO donor SNP， NO scavenger PTIO， nitric oxide synthase（NOS） inhibitor L-NAME， EBR+PTIO and EBR+L-NAME respectively， and the effects of the above treatments on chilling tolerance， reactive oxygen species（ROS） levels and antioxidant defense system were analyzed in the cells under low temperature stress. The results showed that： （1）exogenous EBR treatment enhanced chilling tolerance in the suspension cultures and alleviated the inhibition of cell viability and aggravations of ion leakage and membrane lipid peroxidation induced by low temperature. The effects of SNP treatment on the above physiological measures were similar to those of EBR. （2）Application of PTIO or L-NAME in combination with EBR significantly decreased cell viability and increased ion leakage and membrane lipid peroxidation in C. bungeana suspension cultures under chilling stress compared with those of EBR treatment alone， suggesting that the block in NO signaling decreased the EBR-enhanced chilling tolerance. （3）EBR treatment further increased NO production and NOS activity in the suspension cells compared with those under chilling stress alone， whereas the EBR-induced NO signal was quenched by the addition of PTIO or L-NAME. （4）Both EBR and SNP obviously inhibited the increases in hydrogen peroxide（H₂O₂） content， superoxide radical（{\mathrm{O}}_{\mathrm{2}}^{·–}） production rate and hydroxyl radical （OH^-）content caused by chilling， and remarkably enhanced the activities of ascorbate peroxidase（APX）， catalase（CAT）， glutathione reductase（GR）， peroxidase（POD） and superoxide dismutase（SOD） and contents of ascorbate （AsA） and glutathione（GSH） in the suspension cultured cells， thus alleviating oxidative injury caused by low temperature. However， PTIO and L-NAME blocked the protective effects of EBR. In conclusion， these results suggested that EBR-induced chilling tolerance in C. bungeana suspension cultured cells was through the promotion of NO accumulation by activating NOS activity. EBR might confer an increased tolerance to chilling stress by suppressing the accumulation of ROS caused by chilling and enhancing antioxidant defense system in the suspension cells， both of which were partially regulated by NO signal， resulting in the alleviation of chilling-induced oxidative damage and membrane lipid peroxidation. Thus， NOS-derived NO might be a downstream signaling molecule of EBR signal in C. bungeana suspension cultured cells under low temperature stress.

关键词

油菜素内酯 / 一氧化氮 / 高山离子芥 / 抗寒性 / 活性氧 / 抗氧化防御系统

Key words

brassinosteroids / nitric oxide / Chorispora bungeana / chilling tolerance / reactive oxygen species / antioxidant defense system

Author summay

刘亚洁（1981—），女，实验师，博士，主要从事种子检验及植物逆境生理学研究。