In plants,genetically encoded probes based on redox-sensitive green fluorescent protein(roGFP)have been used to detect hydrogen peroxide(H_(2)O_(2))levels by fusing exogenous thiol peroxidases,such as Orp1 and Tsa2.Ho...In plants,genetically encoded probes based on redox-sensitive green fluorescent protein(roGFP)have been used to detect hydrogen peroxide(H_(2)O_(2))levels by fusing exogenous thiol peroxidases,such as Orp1 and Tsa2.However,the effectiveness of these thiol peroxidases compared to endogenous ones remains unexplored.Here,we develop a H_(2)O_(2) probe by fusing roGFP2 to an endogenous H_(2)O_(2) sensor,type Ⅱ peroxiredoxin(PRXⅡB),which displayed enhanced responsiveness and conversion kinetics compared to roGFP2-Orp1 in vitro and superior sensitivity to H_(2)O_(2) in vivo.The roGFP2-PRXⅡB probe allowed robust visualization of H_(2)O_(2) production in abiotic and biotic stresses,and growing pollen tubes.We further targeted roGFP2-PRXⅡB to cytosol,nuclei,mitochondria and chloroplasts to monitor H_(2)O_(2) accumulation in real time in different subcellular compartments during immune activation,and the analyses revealed different temporal patterns of H_(2)O_(2) accumulation during pattern-and effector-triggered immune responses in different compartments.Taken together,the work provides an ultra-sensitive probe for H_(2)O_(2) dynamics in diverse plant biological processes.展开更多
基金supported by the National Natural Science Foundation of China (32330056 and 32120103004 to J.-M.Z., 32170288 to G.B.)
文摘In plants,genetically encoded probes based on redox-sensitive green fluorescent protein(roGFP)have been used to detect hydrogen peroxide(H_(2)O_(2))levels by fusing exogenous thiol peroxidases,such as Orp1 and Tsa2.However,the effectiveness of these thiol peroxidases compared to endogenous ones remains unexplored.Here,we develop a H_(2)O_(2) probe by fusing roGFP2 to an endogenous H_(2)O_(2) sensor,type Ⅱ peroxiredoxin(PRXⅡB),which displayed enhanced responsiveness and conversion kinetics compared to roGFP2-Orp1 in vitro and superior sensitivity to H_(2)O_(2) in vivo.The roGFP2-PRXⅡB probe allowed robust visualization of H_(2)O_(2) production in abiotic and biotic stresses,and growing pollen tubes.We further targeted roGFP2-PRXⅡB to cytosol,nuclei,mitochondria and chloroplasts to monitor H_(2)O_(2) accumulation in real time in different subcellular compartments during immune activation,and the analyses revealed different temporal patterns of H_(2)O_(2) accumulation during pattern-and effector-triggered immune responses in different compartments.Taken together,the work provides an ultra-sensitive probe for H_(2)O_(2) dynamics in diverse plant biological processes.
