Phosphoproteomic Analysis Of Puccinellia Tenuiflora Leaves And Chloroplasts In Response To Na₂CO₃

Alkali-salinity is one of the most significant abiotic stresses and it limits the productivity and geographical distribution of plants.Alkali-salinity stress not only exerts the osmotic stress and ion injury,but also with the additional influence of high-pH stress.Puccinellia tenuiflora is a halophytic species belonging to the Gramineae,and capable of surviving in highly alkaline soil(e.g.,pH 10).Therefore,P.tenuiflora is considered as an ideal model plant for studying the saline-alkali tolerant mechanisms.Previous studies have generally emphasized the physiological and ecological analysis of P.tenuiflora in response to neutral salt,while little attention has been given to alkali stress.In this study,we investigated the Na2CO3-responsive characteristics in P.tenuiflora chloroplasts using quantitative proteomic and physiological approaches.We analyzed the rapid chlorophyll fluorescence induction kinetics,reactive oxygen species(ROS)scavenging pathway,leaf protein abundance change pattern,reversible phosphorylation modification of leaf and chloroplast protein,and the homologous gene expression level of the chloroplast phosphoproteins in P.tenuiflora under Na2CO3 stress.We found 104 significant abundance changed proteins from the leaf proteome,most of these proteins were predicted localized in chloroplasts.A total of 84 chloroplast phosphoproteins were identified from the leaf and chloroplast phosphoproteome using isobaric tags for relative and absolute quantification(iTRAQ)and stable isotope dimethyl labeling proteomic approaches.The abundance and post-translational modification patterns of these proteins highlight a series of molecular and physiological strategies of P.tenuiflora in response to Na2CO3 stress,such as thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photodamaged photosystem(PS)?,alteration of PS ? activity,and ROS homeostasis.Moreover,a total of 28 chloroplast phosphoprotein homologous genes were analyzed using the real-time quantative PCR(qRT-PCR)method,we found 10 homologous genes were down-regulated,3 homologous genes were stable expression,and 15 homologous genes were up-regulated,which are crucial for the regulation of photosynthesis,Na+ transport,and Ca2+ signaling.Our results indicate that P.tenuiflora leaves and chloroplasts response to Na2CO3 stress at the level of protein abundance,post-translational modification,and gene expression,which have improved our understanding of the Na2CO3-responsive strategies:include(1)maintain the energetic balance between two photosystems and prevent excess photodamage to PS ? through the regulation of PS?-LHC? complexes migration,reorganisation,and reversibly phosphorylation;(2)the enhancement of cyclic electron flow around photosystem ? is essential for photosynthesis under stress;(3)photorespiration pathway is necessary to alleviate the over-reduction of electron transport chain;(4)reduce excessive light energy absorption by decreasing chlorophyll content and prevent the accumulation of intermediates of chlorophyll biosynthesis;(5)minimize the oxidative injury by elevating the specific ROS scavenging pathways;(6)Na+ compartmentalization and considerable accumulation of glycine betaine to maintain the ionic and osmotic homeostasis;(7)dynamics regulation of chloroplast thylakoids architecture alleviate damage to photosynthesis machinery;(8)coordinated synthesis,assembly,and degradation of both the nuclear-and chloroplast-encoded proteins.