Na2CO3-Responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Abstract

4Alkali-salinity exerts severe osmotic, ionic and high-pH stresses to plants. To understand the 6 5 alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive 6 6 oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics 6 7 analyses of alkaligrass (Puccinellia tenuiflora) under Na 2 CO 3 stress were conducted. In 6 8 addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the 6 9 proteomic results and test the functions of the Na 2 CO 3 -responsive proteins. A total of 104 and 7 0 102 Na 2 CO 3 -responsive proteins were identified in leaves and chloroplasts, respectively. In 7 1 addition, 84 Na 2 CO 3 -responsive phosphoproteins were identified, including 56 new 7 2 phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the 7 3 regulation of photosynthesis, ion transport, signal transduction and energy homeostasis. A 7 4 full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) 7 5 was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, 7 6leading to enhanced Na 2 CO 3 tolerance. All these results indicate that thermal dissipation, 7 7 state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI 7 8 activity, and ROS homeostasis were altered in response to Na 2 CO 3 stress, and they have 7 9improved our understanding of the Na 2 CO 3 -responsive mechanisms in halophytes. 8 0 8 1 8 2 Puccinellia tenuiflora 8 3 8 4 8 7 most severe abiotic stresses, limiting the productivity and geographical distribution of plants. 8 8Saline-alkali stress exerts osmotic stress and ion damage, as well as high-pH stress to plants 8 9[2]. However, little attention has been given to the sophisticated tolerance mechanisms 9 0 underlying plant response to saline-alkali (e.g. Na 2 CO 3 and NaHCO 3 ) stresses [3,4]. As the 9 1 organelle for photosynthesis, chloroplasts are extremely susceptible to saline-alkali stress [5]. 2Excessive accumulation of Na + reduces the CO 2 diffusion through stomata and mesophyll, 9 3 negatively affecting plant photosynthesis [6]. As a consequence, excessive excitation energy 9 4 causes generation of reactive oxygen species (ROS), resulting in damage to the thylakoid 9 5 membrane [6]. 9 6 4 Current high-throughput proteomic approaches are powerful to untangle the complicated 9 7 mechanisms of chloroplast development, metabolism and stress response [7−10]. More than 9 8 522 NaCl-responsive chloroplast proteins were found in different plant species, such as 9 9 tomato (Solanum lycopersicum) [11], wheat (Triticum aestivum) [12], and other plant species 1 0 0 [13−18]. The presence of these proteins indicate that the light harvesting, photosynthetic 1 0 1 electron transfer, carbon assimilation, ROS homeostasis, energy metabolism, signaling, and 4 9 5

NaCl-responsive ROS scavenging and energy supply in alkaligrass callus revealed from proteomic analysis

“…In the NaCl-stressed alkaligrass calli, the glycine betaine content was significantly increased. This result was consistent with the results from NaCl-stressed sugarcane calli [13] and the salinity-or alkali treated alkaligrass leaves [32,35,36].…”

“…In the alkaligrass callus, the activities of APX, MDHAR and GR were NaCl-increased, but DHAR activity was NaCl-decreased. This result was similar to that in leaves under Na 2 CO 3 -treatmenf for 12h and 24h [35]. In addition, the amount of APX and an isoform of MDHAR, as well as the AsA content were all increased in the alkaligrass callus after salt stress.…”

“…CAT pathway can directly dismutase H 2 O 2 to H 2 O and O 2 in peroxisomes. In salinity-/alkali-stressed alkaligrass, CAT activities were decreased in the calli and short-term (12h and 24h) salt-treated leaves and roots [34,35], but increased in long-term (seven days) salt-treated leaves [32,33]. POD also catalyzes the reduction of H 2 O 2 using various substrates, such as phenolic compounds, lignin precursors, auxin and secondary metabolites.…”

NaCl-responsive ROS scavenging and energy supply in alkaligrass callus revealed from proteomic analysis

“…CAT pathway can directly dismutase H 2 O 2 to H 2 O and O 2 in peroxisomes. In salinity-/alkali-stressed alkaligrass, CAT activities were reduced in callus and short-term (12h and 24h) salt-treated leaves and roots [32,33], but induced in long-term (7 days) salt-treated leaves [30,31]. POD also catalyzes the reduction of H 2 O 2 using various substrates, such as phenolic compounds, lignin precursors, auxin and secondary metabolites.…”

“…Alkaligrass (Puccinellia tenuifora) is a monocotyledonous halophyte with high salinity and alkali tolerance. Our previous proteomics and physiological studies have reported the salt-/alkali-responsive mechanisms in leaves and roots in response to NaCl [30], Na 2 CO 3 [31][32][33], and NaHCO 3 [34] stresses.…”