Full Text:   <445>

Summary:  <138>

Suppl. Mater.: 

CLC number: S432.1

On-line Access: 2019-08-05

Received: 2019-03-01

Revision Accepted: 2019-05-12

Crosschecked: 2019-07-09

Cited: 0

Clicked: 2302

Citations:  Bibtex RefMan EndNote GB/T7714


Yan Liang


-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2019 Vol.20 No.9 P.713-727


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

Author(s):  Qiu-Ying Li, Ping Li, Nang Myint Phyu Sin Htwe, Ke-Ke Shangguan, Yan Liang

Affiliation(s):  Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insects, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China

Corresponding email(s):   yanliang@zju.edu.cn

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

Qiu-Ying Li, Ping Li, Nang Myint Phyu Sin Htwe, Ke-Ke Shangguan, Yan Liang. 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]. Journal of Zhejiang University Science B, 2019, 20(9): 713-727.

@article{title="Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis",
author="Qiu-Ying Li, Ping Li, Nang Myint Phyu Sin Htwe, Ke-Ke Shangguan, Yan Liang",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis
%A Qiu-Ying Li
%A Ping Li
%A Nang Myint Phyu Sin Htwe
%A Ke-Ke Shangguan
%A Yan Liang
%J Journal of Zhejiang University SCIENCE B
%V 20
%N 9
%P 713-727
%@ 1673-1581
%D 2019
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1900105

T1 - Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis
A1 - Qiu-Ying Li
A1 - Ping Li
A1 - Nang Myint Phyu Sin Htwe
A1 - Ke-Ke Shangguan
A1 - Yan Liang
J0 - Journal of Zhejiang University Science B
VL - 20
IS - 9
SP - 713
EP - 727
%@ 1673-1581
Y1 - 2019
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1900105

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.




Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Antolín-Llovera M, Ried MK, Binder A, et al., 2012. Receptor kinase signaling pathways in plant‒microbe interactions. Annu Rev Phytopathol, 50:451-473.

[2]Baker CJ, Orlandi EW, 1995. Active oxygen in plant pathogenesis. Annu Rev Phytopathol, 33:299-321.

[3]Bedard K, Krause KH, 2007. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 87(1):245-313.

[4]Bedard K, Lardy B, Krause KH, 2007. NOX family NADPH oxidases: not just in mammals. Biochimie, 89(9):1107-1112.

[5]Boller T, Felix G, 2009. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol, 60:379-406.

[6]Calcaterra NB, Pico GA, Orellano EG, et al., 1995. Contribution of the FAD binding site residue tyrosine 308 to the stability of pea ferredoxin-NADP+ oxidoreductase. Biochemistry, 34(39):12842-12848.

[7]Cao YR, Liang Y, Tanaka K, et al., 2014. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife, 3: e03766.

[8]Chen DQ, Cao YR, Li H, et al., 2017. Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture. Nat Commun, 8(1):2265.

[9]Chen F, Haigh S, Yu YF, et al., 2015. Nox5 stability and superoxide production is regulated by C-terminal binding of Hsp90 and CO-chaperones. Free Radical Biol Med, 89:793-805.

[10]Chinchilla D, Zipfel C, Robatzek S, et al., 2007. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature, 448(7152):497-500.

[11]Choi J, Tanaka K, Liang Y, et al., 2014. Extracellular ATP, a danger signal, is recognized by DORN1 in Arabidopsis. Biochem J, 463(3):429-437.

[12]Debeurme F, Picciocchi A, Dagher MC, et al., 2010. Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly. J Biol Chem, 285(43):33197-33208.

[13]Dodds PN, Rathjen JP, 2010. Plant immunity: towards an integrated view of plant–pathogen interactions. Nat Rev Genet, 11(8):539-548.

[14]Doke N, 1983. Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiol Plant Pathol, 23(3):345-357.

[15]Dubiella U, Seybold H, Durian G, et al., 2013. Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation. Proc Natl Acad Sci USA, 110(21):8744-8749.

[16]Heyworth PG, Cross AR, Curnutte JT, 2003. Chronic granulomatous disease. Curr Opin Immunol, 15(5):578-584.

[17]Kadota Y, Sklenar J, Derbyshire P, et al., 2014. Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell, 54(1):43-55.

[18]Kadota Y, Liebrand TWH, Goto Y, et al., 2019. Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector- and PAMP-triggered immunity in plants. New Phytol, 221(4):2160-2175.

[19]Kawahara T, Quinn MT, Lambeth JD, 2007. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol Biol, 7:109.

[20]Kawahara T, Jackson HM, Smith SM, et al., 2011. Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain. Biochemistry, 50(12):2013-2025.

[21]Knight H, Knight MR, 1995. Recombinant aequorin methods for intracellular calcium measurement in plants. Methods Cell Biol, 49:201-216.

[22]Kuhns DB, Alvord WG, Heller T, et al., 2010. Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med, 363(27):2600-2610.


[24]Lamb C, Dixon RA, 1997. The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol, 48: 251-275.

[25]Lambeth JD, 2004. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol, 4(3):181-189.

[26]Leben R, Ostendorf L, van Koppen S, et al., 2018. Phasor-based endogenous NAD(P)H fluorescence lifetime imaging unravels specific enzymatic activity of neutrophil granulocytes preceding NETosis. Int J Mol Sci, 19(4):1018.

[27]Leto TL, Morand S, Hurt D, et al., 2009. Targeting and regulation of reactive oxygen species generation by Nox family NADPH oxidases. Antioxid Redox Signal, 11(10):2607-2619.

[28]Li L, Li M, Yu LP, et al., 2014. The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe, 15(3):329-338.

[29]Liang Y, Cao YR, Tanaka K, et al., 2013. Nonlegumes respond to rhizobial Nod factors by suppressing the innate immune response. Science, 341(6152):1384-1387.


[31]Liao DH, Cao YR, Sun X, et al., 2017. Arabidopsis E3 ubiquitin ligase PLANT U-BOX13 (PUB13) regulates chitin receptor LYSIN MOTIF RECEPTOR KINASE5 (LYK5) protein abundance. New Phytol, 214(4):1646-1656.

[32]Liu YK, He CZ, 2016. Regulation of plant reactive oxygen species (ROS) in stress responses: learning from AtRBOHD. Plant Cell Rep, 35(5):995-1007.

[33]Macho AP, Zipfel C, 2014. Plant PRRs and the activation of innate immune signaling. Mol Cell, 54(2):263-272.

[34]Magnani F, Nenci S, Fananas EM, et al., 2017. Crystal structures and atomic model of NADPH oxidase. Proc Natl Acad Sci USA, 114(26):6764-6769.

[35]Marino D, Dunand C, Puppo A, et al., 2012. A burst of plant NADPH oxidases. Trends Plant Sci, 17(1):9-15.

[36]Mehdy MC, 1994. Active oxygen species in plant defense against pathogens. Plant Physiol, 105(2):467-472.

[37]Melotto M, Underwood W, Koczan J, et al., 2006. Plant stomata function in innate immunity against bacterial invasion. Cell, 126(5):969-980.

[38]Mittler R, Vanderauwera S, Suzuki N, et al., 2011. ROS signaling: the new wave? Trends Plant Sci, 16(6):300-309.

[39]Niesner R, Narang P, Spiecker H, et al., 2008. Selective detection of NADPH oxidase in polymorphonuclear cells by means of NAD(P)H-based fluorescence lifetime imaging. J Biophys, 2008, 2008:602639.

[40]Oda T, Hashimoto H, Kuwabara N, et al., 2010. Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications. J Biol Chem, 285(2):1435-1445.

[41]Ogasawara Y, Kaya H, Hiraoka G, et al., 2008. Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J Biol Chem, 283(14):8885-8892.

[42]Peccarelli M, Kebaara BW, 2014. Regulation of natural mRNAs by the nonsense-mediated mRNA decay pathway. Eukaryot Cell, 13(9):1126-1135.

[43]Qi JS, Wang JL, Gong ZZ, et al., 2017. Apoplastic ROS signaling in plant immunity. Curr Opin Plant Biol, 38: 92-100.

[44]Segal BH, Grimm MJ, Khan ANH, et al., 2012. Regulation of innate immunity by NADPH oxidase. Free Radical Biol Med, 53(1):72-80.

[45]Shang-Guan K, Wang M, Htwe NMPS, et al., 2018. Lipopolysaccharides trigger two successive bursts of reactive oxygen species at distinct cellular locations. Plant Physiol, 176(3):2543-2556.

[46]Singel KL, Segal BH, 2016. NOX2-dependent regulation of inflammation. Clin Sci, 130(7):479-490.

[47]Stasia MJ, Li XJ, 2008. Genetics and immunopathology of chronic granulomatous disease. Semin Immunopathol, 30(3):209-235.

[48]Sumimoto H, 2008. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J, 275(13):3249-3277.

[49]Suzuki N, Miller G, Morales J, et al., 2011. Respiratory burst oxidases: the engines of ROS signaling. Curr Opin Plant Biol, 14(6):691-699.

[50]Suzuki N, Koussevitzky S, Mittler R, et al., 2012. ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ, 35(2):259-270.

[51]Torres MA, Dangl JL, Jones JDG, 2002. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA, 99(1):517-522.

[52]Verhoeven AJ, 1997. The NADPH oxidase: lessons from chronic granulomatous disease neutrophils. Ann N Y Acad Sci, 832:85-92.

[53]Vignais PV, 2002. The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell Mol Life Sci, 59(9):1428-1459.

[54]Zhang MX, Chiang YH, Toruño TY, et al., 2018. The MAP4 kinase SIK1 ensures robust extracellular ROS burst and antibacterial immunity in plants. Cell Host Microbe, 24(3):379-391.e5.

[55]Zhang YY, Zhu HY, Zhang Q, et al., 2009. Phospholipase Dα1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. Plant Cell, 21(8):2357-2377.

[56]Zhang ZJ, Peck SC, 2011. Simplified enrichment of plasma membrane proteins for proteomic analyses in Arabidopsis thaliana. Proteomics, 11(9):1780-1788.

[57]List of electronic supplementary materials

[58]Table S1 Primer sequences used in this study

[59]Fig. S1 Allelic mutants delt1-1 and delt1-2

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - Journal of Zhejiang University-SCIENCE