The Mechanism Of Development-dependent H₂O₂ Accumulation In Maize Subsidiary Cells

Stomata are microscopic pores that exist in most aerial tissues of higher plants.Plants can accurately respond to developmental and environmental changes by regulating actively stomatal movement,thus optimizing water use efficiency and photosynthetic activity.The signaling molecular mechanism that H₂O₂ in guard cells produced by the apoplast pathway is involved in regulating stomatal movement,has been deeply analyzed,little is known about the production of H₂O₂ and mechanism of stomatal movement in subsidiary cells of grass stomata.This study focused on the developmental-dependent H₂O₂ accumulation in subsidiary cells,using wild-type maize variety Zhengdan 958 and ABA synthesis mutant vp14 as experimental materials,and different leaf ages?3th-leaf age,5th-leaf age and 7th-leaf age?for treatments.The ABA content,leave relative water content,photosynthetic activity and wax content of functional leaves were determined under different leave ages and drought stress,with the methods of physiology,pharmacology and molecular genetics,demonstrating that development-dependent H₂O₂ accumulation in subsidiary cells was related with ABA level variation.Next,the sources and transport mechanism of H₂O₂ in subsidiary cells were anylased,which provided theoretical basis for furtherly investigating how developmental-related H₂O₂ regulates stomatal movement in subsidiary cells.The results and conclusions were as followings:1.Development-dependent H₂O₂ accumulation in subsidiary cells was related with ABA level variation:Under normal water conditions,with the increase of leaf ages,namely,from 3th-leaf age,5th-leaf age to 7th-leaf age,ABA content,wax content,photosynthetic activity of functional leaves increased,while leave relative water content decreased;treatment epidermis strips of vp14 7th-leaf age and Zhengdan 958 3th-leave age with exogenous 100?M ABA,H₂O₂accumulation was found in subsidiary cells;light-dark transition for vp14,or pretreatment Zhengdan 958 with 10?M Flu then light-dark transition,there were no accumulation of H₂O₂in subsidiary cells.Under drought stress,leave relative water content of functional leaves in3th-leaf age decreased,while,ABA content,photosynthetic activity and wax content increase.Besides,there was H₂O₂ accumulation in subsidiary cells.2.Development-dependent H₂O₂ accumulation in subsidiary cells was related with NADPH oxidase:Under normal water conditions,with the increase of leaf ages,NADPH oxidase activity,NADPH content and transcription level of ZmRBOHD,ZmRBOHFR in functional leaves increased,while both PAO activity and Spd content decreased,Spm content unchanged;Pretreatment epidermis strips of Zhengdan 958 with NADPH oxidase and PAO inhibitors,namely 10?M DPI and 1mM AG,respectively,and then treatment epidermis strips of3th-leave with exogenous ABA,or direct treatment epidermis strips of 7th-leave for light-dark conversion,there were no accumulation of H₂O₂ in subsidiary cells;treatment epidermis strips of Zhengdan 958 3th-leave age with exogenous 1mM Spd,H₂O₂ accumulation was observed in subsidiary cells;while treatment the epidermis strips with exogenous 1mM,10mM,20mM Spm,there was no accumulation of H₂O₂ in subsidiary cells.Under drought stress,NADPH oxidase activity,NADPH content,and transcription level of ZmRBOHD,ZmRBOHFR increased in the functional leaves of the 3th-leaf stage,which corresponds to the accumulation of H₂O₂ in the subsidiary cells.3.Changes of PIP aquaporin gene transcription level with the development of leave ages:Under normal water conditions,transcription level of ZmPIP2;1,ZmPIP2;2,and ZmPIP2;5 in functional leaves significantly increased with leaf ages increase.Under drought stress,transcription level of functional leaves ZmPIP2;1,ZmPIP2;2,and ZmPIP2;5 were significantly up-regulated,corresponding to the accumulation of H₂O₂ in subsidiary cells.In conclusion,this study demonstrated that ABA was involved in the development-dependent of H₂O₂ accumulation in subsidiary cells from both physiological and molecular genetic levels.Here,ABA was an indication of leaf tissue water status.Under normal water conditions,compared with three-leaf age,seedlings of 7th-leave age might have strong metabolism and transpiration,and reduced relative water content of leaves,resulting in a significant increase in ABA synthesis and induction of H₂O₂ accumulation in subsidiary cells.Drought stress induced the decrease of leave relative water content in functional leaves of 3th-leave age,increased ABA synthesis,and accumulation of H₂O₂ in subsidiary cells.In both cases,H₂O₂ in subsidiary cells increased the efficiency and plasticity of stomatal regulation to avoid damage to plants caused by low water potential.At the same time,it hadbeen confirmed from the physiological level that the production of H₂O₂ in development-dependent subsidiary cells may originate from NADPH oxidase,but this needed furtherly to be studied from the molecular genetic level.