Our recently published results demonstrated a crucial role for plastid terminal oxidase (PTOX) as an alternative electron pathway in the halophyte Spartina alterniflora (S. alterniflora) under salt stress but not for the glycophyte Setaria viridis (S. viridis). Herein, the effect of salt on the photosynthetic electron transport and RNA-seq analysis was probed in Setaria and its salt-tolerant close relative S. alterniflora. Initially, plants were grown at soil then were salt-treated under hydroponic conditions for two weeks. Setaria shows high vulnerability to salt compared to Spartina; while, Setaria was unable to survive exposure to greater than 100 mM, Spartina could tolerate salt concentrations as high as 550 mM with merely negligible effect on gas exchange and conductance of electrons transport chain (gETC). After exposure to salt, the prompt fluorescence (OJIP-curves) reveals an increase in the O- and J-steps in Setaria and very less or no change for SA. This suggests a higher QA over-reduction in Setaria than in Spartina. Following salt treatment, a dramatic decline in PSII primary photochemistry for Setaria was observed, as displayed by the drastic drop in Fv/Fm, Fv/Fo and ΦPSII. However, no substantial change was recorded regarding these parameters for Spartina under NaCl treatment. Interestingly, we report an improvement in primary PSII photochemistry (ΦPSII) for Spartina with increasing either salt concentration or duration. Besides, the magnitude of NPQ dynamics was strongly enhanced for Setaria even at low NaCl level (50 mM); however, it remains unchangeable or slightly increased for Spartina at high NaCl concentrations (above 400 mM). For plants endured salt, we notice an increase in both the proportion of oxidized P700 and the amount of active P700 in Setaria and almost no change for Spartina. The slowdown of electrons flow through PSII was accompanied by a dramatic decline in gETC. Under salt, CO2 assimilation (A) and stomatal conductance (gs) evaluations demonstrate that A decreases earlier, even after one week exposure to only 50 mM NaCl for Setaria; however, the effect of salt was negligible in Spartina regarding these two parameters even after exposure for two weeks to high salt levels (400 and 550 mM). For Setaria exposed for 12 d to salt, the use of 2,000 μmol m-2 s-1 external CO2 was not sufficient to fully restore A to the control level as assessed by A-Ci curves, even for 50 mM salt. The A at all NaCl levels, except 550 mM, was able to completely recover to initial level before stress in Spartina. RNAseq analysis shows a stimulation of oxido-reduction reactions in Setaria. Gene onthology (GO) enrichment emphasizes differentially expressed genes (DEGs) and some transcription factors (TFs) under salt. The up-regulated genes in Setaria are related to three metabolic processes; C4, photorespiration and the oxidation/reduction pathways. Some other specifically highly up-regulated genes in Setaria are mostly related to TFs including DNA-binding transcription factor activity, stress marker genes such as peroxidase and senescence-related genes such as flavonol synthase.
Overall design: Seeds of Spartina (thereafter S. alterniflora) were collected from San-San Lake in South East Shanghai city at november end (2016 and 2017). The cleaned spikelets were stored in wet tissue in sealed plastic in the refrigerator at 4°C. S. alterniflora seeds require between two and three months after ripening in cold, wet storage (stratification) to break dormancy (Garbisch and McIninch, 1992) and they remain viable for about one year. Setaria viridis (thereafter S. viridis) grass seeds were stored in a dry place. Seeds of Spartina were rinsed several times with tap water outdoors to reduce odors that come out then transfer them to Petri-dishes and covered with water till germinate then transfer them to potted soil. When the young seedlings of Spartina are about 2 cm in length and started turning green, they then removed from the glass petri dishes and seeds. Trays containing Spartina seedlings were kept indoor at a temperature controlled room of (25-27°C), under fluorescent grow light (80 - 120 μmol m-2 s-1) on a photoperiod of 16/8 hour (light/dark) photoperiod. Then two-month old healthy plants with large expanded leaves where transferred to hydroponic for salt treatment.
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