活性氧物质的产生通常是对于生物或非生物胁迫的早期响应。虽然，ROS是动植物中的一个的非常重要的信号物质，但是其不受限制的积累会导致有害的影响，比如说细胞死亡。因此，生物体内ROS的产生是受到严格调控的。胞外ROS产生的一个主要组分就是演化上比较保守的NADPH氧化酶NOX家族。在动植物中，NOX依赖性的ROS产生参与了免疫响应、细胞生长以及凋亡等生物进程的调控（Kimura et al., 2017；Waszczak et al., 2018）。植物中的NOX又叫呼吸爆发氧化酶同源物RBOH，包含六个跨膜螺旋、一个C端的黄素腺嘌呤二核苷酸和NADPH结合结构域，以及一个N端的钙离子结合EF-hand。虽然先前的研究显示，RBOH蛋白N端的磷酸化具有主要作用，但人类NOX蛋白的研究显示其C端的磷酸化对于ROS产量调控非常重要（Raad et al., 2009）。然而，植物中RBOH蛋白的C端是否能够被磷酸化还不清楚。

Extracellular RBOH-dependent ROS production is triggered by the activation of receptor-like protein kinases (RLKs). A large group of RLKs in plants are the Cys-rich RLKs (CRKs), which are important signaling components in plant development and the perception of biotic and abiotic stresses. A new study by Kimura et al. (2020) has found that CRK2 forms a preactivation complex with RBOHD in Arabidopsis (Arabidopsis thaliana) and, importantly, phosphorylates the C terminus of RBOHD in vivo to regulate ROS production.

胞外RBOH依赖性的ROS产量由激活的类受体蛋白激酶RLKs所诱导。植物中，一大类RLKs属于富含半胱氨酸类受体蛋白CRKs，这些激酶是植物发育和感知生物及非生物胁迫信号通路的关键组分。Kimura等人最新的研究显示拟南芥中CRK2与RBOHD形成一个预激活的复合物，并且RBOHD蛋白C端的磷酸化调控ROS产量。

The authors first showed that CRK2 and its kinase activity is important for proper plant growth. Next, they tested the role of CRK2 in regulating ROS production and immune responses. Mutant crk2 plants had reduced ROS production triggered by the microbe-associated molecular pattern (MAMP) flg22. The crk2 mutant was also more susceptible to the bacterial pathogen Pseudomonas syringae. Other MAMP-triggered responses, such as stomatal closure, Ca2+ influx, MAPK gene activation, and early callose deposition, were also altered in crk2.

首先，作者显示了CRK2及其激酶活性对于植物正常的生长是非常重要的。接着，作者进一步测试了CRK2在调控ROS产量和免疫响应方面的功能。crk2突变体植株中，由微生物相关分子模式MAMP诱导产生的ROS明显减少。并且，crk2突变体对于细菌性病原体丁香假单胞菌十分易感。同样，crk2突变体中其它的MAMP诱导响应也被改变，比如气孔闭合、钙离子内流、MAPK基因激活以及早期胼胝质沉积等。

CRK2 interacted with several RBOHs in vitro, while in vivo RBOHD coimmunoprecipitated with CRK2 with or without flg22 treatment, suggesting that CRK2 preassociates with RBOHD independent of flg22 perception. This is in contrast to many other RLKs that only form complexes in response to ligand binding.

体外试验中，CRK2能够与一些RBOHs互作；但在体内试验中，无论有没有flg22处理，RBOHD都能够与CRK2免疫功沉淀，说明CRK2独立于感知flg22而与RBOHD提前相互关联。这与其它的RLKs不同，其它RLKs一般只有在响应配体结合之后才会形成复合物。

Although CRK2 phosphorylated RBOHD at both the N and C termini in vitro, phosphorylation at the C terminus was shown to be important for regulation of RBOHD activity by CRK2. Phosphorylation of the C-terminal residue Ser-703 (S703) was enhanced upon flg22 treatment and a dead kinase version of CRK2 significantly reduced phosphorylation of S703 in planta, suggesting that CRK2 and its kinase activity are at least partially responsible for flg22-induced phosphorylation of S703. ROS production and stomatal closure triggered by flg22 were reduced in RBOHDS703A plants, in which S703 was replaced with Ala. The RBOHDS703A plants were more susceptible to the pathogen P. syringae. Thus, phosphorylation of S703 of RBOHD is important for regulating plant immune responses.

尽管在体外，CRK2能够同时磷酸化RBOHD的C端和N端，但是RBOHD蛋白C端的磷酸化对于由CRK2介导的RBOHD活性调控是非常重要的。通过flg22的处理，可以增强RBOHD蛋白C端Ser-703位点的磷酸化，并且CRK2激酶活性缺失会导致S703位点的磷酸化水平显著降低，说明CRK2及其激酶活性至少部分参与了由flg22诱导的RBOHD蛋白C端S703磷酸化。另外，在703号位点丝氨酸被替换为不能被磷酸化的丙氨酸的RBOHDS703A植株中，由flg22诱导的ROS产量和气孔闭合都明显减少。并且，RBOHDS703A植株对于丁香假单胞菌表现出易感表型。因此，RBOHD蛋白的S703磷酸化对于植物免疫响应的调控是非常重要的。

The phospho sites in the RBOHD C terminus displayed strong conservation throughout the entire plant RBOH clade and even in human NOX2 and NOX5, suggesting strong evolutionary constraints to preserve the functions of phosphorylation at the C terminus.

RBOHD蛋白C端的磷酸化位点在整个植物RBOH类中都十分保守，并且与人类的NOX2和NOX5显示出保守性，说明存在一个强大的演化限制，以保证该NADPH氧化酶C端位点的磷酸化发挥其功能。

Based on these results, the authors propose that regulating NADPH oxidase activity by phosphorylation at the C terminus might be an ancient mechanism that coordinates with other mechanisms targeting the N terminus (see figure). The diverse processes that activate RBOHs indicate a complex network strictly controlling ROS production and its signaling function.

基于以上的结果，作者提出通过C端磷酸化调控NADPH氧化酶活性可能是一个比较古老的机制，与其它N端调控机制一起协调发挥作用。不同的RBOH激活机制组成了一个复杂的网络，严格限制了ROS产量及其信号转导功能。