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Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis

拟南芥 NADPH 氧化酶 RBOHD 羧基端倒数第三位氨基酸在其介导活性氧迸发中的重要作用

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Abstract

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.

抽象

目 的

解析呼吸爆发氧化酶同系物蛋白 D (RBOHD) 介导活性氧迸发的分子机制。

创新点

首次研究 RBOHD 蛋白羧基端在植物体内活性氧迸发中的作用, 并对其机制进行初步探究。

方法

本文利用正向遗传学方法筛选得到在多种病原物相关分子模式 (PAMP) 处理后活性氧不迸发的突变体delt。然后结合图位克隆和全基因测序技术, 发现 DELT1 编码了 RBOHD 蛋白。DELT1-2 在 RBOHD 羧基端倒数第三位谷氨酸位置发生了突变。深入分析发现, 谷氨酸的突变不影响 DELT1-2 表达、蛋白定位和互作等功能, 但会导致植物不响应 PAMP 诱导的气孔关闭。

结 论

RBOHD 羧基端倒数第三位谷氨酸对其功能发挥起着决定作用。

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Acknowledgments

We thank Dr. Kun JIANG (College of Life Science, Zhejiang University, Hangzhou, China) for the instruction of the stomatal closure assay.

Author information

Correspondence to Yan Liang.

Additional information

Project supported by the National Natural Science Foundation of China (No. 31622006) and the Postdoctoral Science Foundation of China (Nos. 2018M630683 and 2018T110601)

Contributors

Yan LIANG planned and designed the research; Qiu-ying LI and Ping LI performed most of the experiments; Nang MYINT PHYU SIN HTWE and Ke-ke SHANGGUAN contributed to the cloning of DELT1. Qiu-ying LI and Ping LI wrote the draft manuscript and Yan LIANG revised the manuscript. All authors read and approved the final manuscript. Therefore, all authors have full access to all the data in the study and take responsibility for the integrity and security of the data.

Compliance with ethics guidelines

Qiu-ying LI, Ping LI, Nang MYINT PHYU SIN HTWE, Ke-ke SHANGGUAN, and Yan LIANG declare that they have no conflict of interest.

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Li, Q., Li, P., Htwe, N.M.P.S. et al. Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis. J. Zhejiang Univ. Sci. B 20, 713–727 (2019). https://doi.org/10.1631/jzus.B1900105

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Key words

  • Reactive oxygen species (ROS)
  • NADPH oxidase (NOX)
  • Microbe associated molecular pattern (MAMP)
  • Lipopolysaccharides (LPS)
  • Respiratory burst oxidase homolog D (RBOHD)

CLC number

  • S432.1

关键词

  • 活性氧
  • NADPH 氧化酶
  • 微生物相关分子模式
  • 脂多糖
  • 呼吸爆发氧化酶同系物蛋白 D (RBOHD)