MAPK级联途径参与苹果砧木缺铁应答ROS信号通路的研究

Iron is one of the important factors that influence growth and fruit production. Although it has a realistic meaning to reveal the regulatory mechanism of iron absorption in apple rootstock, the process is still not clear. Our study found that Reactive oxygen species (ROS) in apoplast as the regulatory factor response iron deficiency and the main source of ROS is NADPH oxidase (NOX) located in plasma membrane and regulated by RBOH in Malus xiaojinensis which is the first apple genotype identified as being resistant to iron deficiency; however, MAPK cascade system could involve in ROS signal transduction. Based on the above content, in this study two kinds of apple rootstocks (tolerance/intolerance) will be used to detect physiological indexes under exogenous reagents treatment to confirm ROS signal differences in early iron deficiency stress. Meanwhile, RBOH6 gene that is closely related to iron deficiency stress and MPK3-2/4-1 members screened from MAPKs gene family act as points of the research, and verify the regulation of MAPK protein on NOX using technologies like yeast two-hybrid, BiFC and transgenic technology and identify the relationship with iron deficiency downstream genes in ROS signal transduction. As a consequence, it not only contributes to the difference source of ROS passages between different iron tolerance characteristics in apple rootstock but also clarifies how ROS signaling caused by iron deficiency drive function genes to reply stress through regulating MAPK cascade system, which will provide theoretical bases for revealing the mechanism of iron deficiency resistance in apple rootstock.

铁素是影响苹果树体生长和果实生产的重要因素之一，探究苹果砧木吸铁调控机制有着现实意义，但尚不够清晰。研究发现耐缺铁砧木小金海棠质外体活性氧（ROS）可作为调控因子响应缺铁胁迫，其来源主要是RBOH编码的质膜NADPH氧化酶（NOX）；而MAPK级联系统可参与ROS信号转导。基于此，本研究将同时利用两种苹果砧木（耐/不耐）为材料，通过外施处理，检测生理指标以确定其对于缺铁响应早期的ROS信号差异；并以筛选到的与缺铁胁迫密切相关的RBOH6和MAPKs基因家族成员MPK3-2/4-1为切入点，利用酵母双杂交、BiFC、转基因等技术验证MAPK蛋白对NOX的调控、及在ROS信号通路中与缺铁应答反应下游基因的关系。最终希望阐明不同耐缺铁特性苹果砧木在ROS通路中的差异来源，以及由缺铁胁迫引发的ROS信号如何通过调控MAPK级联途径驱动功能基因应对胁迫，最终为揭示苹果砧木耐缺铁机制提供理论依据。

铁是植物必须的重要微量元素之一，果树在田间种植生产中常受到缺铁胁迫的影响。在铁吸收过程中，活性氧（ROS）可作为信号启动缺铁应答响应，因此研究果树吸铁过程中ROS信号通路的调控机制有着重要意义。本研究中我们以铁高效型苹果砧木小金海棠（Malus xiaojinensis Cheng et Jiang）为主要材料，筛选获得缺铁诱导下调控ROS产生的基因MxRBOH2与MxRBOH6，并通过转基因试验证实二者正向调控苹果的缺铁应答。进而从小金海棠根部特异性表达的蛋白激酶MAPK基因家族成员中，筛选出缺铁处理后表达量显著升高的MxMPK3-2、MxMPK4-1、MxMPK6-2，并证实三者能够在缺铁诱导下响应ROS信号。通过对筛选出的MxMPKs与缺铁相关基因/转录因子的互作筛选，获得一个与MxMPK6-2互作的铁应答关键转录因子MxbHLH104。转基因试验结果表明，缺铁诱导MxMPK6-2在体内磷酸化MxbHLH104，增强转基因苹果愈伤的三价铁还原酶活性与铁吸收通路基因的表达，正向调控缺铁响应，且ROS的存在促进这一过程。MxMPK6-2对MxbHLH104的磷酸化增强了MxbHLH104对MxHA2的转录激活作用。同时，MxMPK6-2也能够磷酸化另一个对MxHA2起转录抑制功能的负调控因子MxERF4，这种磷酸化作用降低了MxERF4对MxHA2的转录抑制功能，与MxMPK6-2-MxbHLH104一起协同调控植株泌氢。此外，也发现MxMPK4-1与MxRBOH2/MxRBOH6存在相互作用，这条途径可能反馈调控缺铁胁迫下ROS信号的产生。.综上，本研究发现了缺铁胁迫下ROS信号调控小金海棠中MAPK级联途径的机制。即缺铁胁迫诱导MxRBOH2/MxRBOH6的表达，产生的ROS信号激活了MxMPK6-2 - MxbHLH104的磷酸化途径，正调控缺铁响应。本研究阐明由缺铁胁迫引发的ROS信号如何通过调控MAPK级联途径驱动功能基因应对胁迫，为揭示苹果砧木耐缺铁机制提供了理论依据。