Novel Insights Into Ser/Thr Protein Kinase SpkI Upon Salt Stress In Synechocystis Sp.PCC 6803

Photosynthesis performed by higher plants,algae and cyanobacteria has built the basis for the emergence of various life forms on earth,and plays a vital role in maintaining the balance of biosphere.However,the photosynthetic efficiency is often challenged by lots of adverse environmental factors.High salinity is one of these common environmental factors,which not only limits the normal growth of these photosynthetic organisms but also affects the stability and functionality of photosynthetic apparatus.Therefore,investigation of the salt adaptation mechanisms of photosynthetic organisms is particularly important for maintaining high-efficient photosynthesis during high salt acclimation.To explore novel genes involved in high salt adaptation mechanisms,a transposon insertion mutant library of unicellular cyanobacteria Synechocystis sp.PCC 6803 was constructed and screened under normal growth condition and high salt condition.Among a number of high salt sensitive mutants obtained,one mutant was selected and identified that the transposon was inserted in the gene sll1770,encoding an ABC1-type Ser/Thr protein kinase SpkI.On the genome,the locus of spkI was adjacent to that of the gene sll1771,encoding a PP2C-type phosphatase PphA.Preliminary screening result suggested that the spkI transposon mutant did not show any obvious difference comparing to WT under control condition,but displayed slower growth rate and decreased maximal quantum yield of PSⅡ under high salt condition.These high salt sensitive phenotypes were confirmed in the insertional kinase mutant Δspkl and were recovered in the complementary strain PpetJ::sll1770.However,the phosphatase mutant ΔpphA did not show any significant differences in growth under both conditions.The photosynthetic activity analysis revealed that there was no significant difference between Δspkl and WT when grown under control condition,while the net photosynthesis rate and PSⅡ activity of ΔspkI was significantly lower than WT and the complementary strain when grown under high salt condition.Immunoblot analysis indicated that the accumulation of major photosynthetic protein subunits,including PSⅡ,PSⅠ,NDH and RbcL,in the mutant ΔspkI was remarkably decreased under high salt condition.All these results suggested that the kinase SpkI plays crucial roles on both the growth and photosynthesis of Synechocystis upon salt stress.Furthermore,the analysis of chlorophyll fluorescence induction and the redox kinetics of photosystem Ⅰ and cytochrome b6f complex showed that the mutation of gene spkI modified the electron transfer of PSⅡ,PSⅠ and cytochrome b6f complex when grown under high salt condition,which caused the slower re-oxidation of QA-,the slower oxidation rate and the faster reduction rate of P700 and cytochrome f as well as plastocyanin,and finally resulted in an altered redox state of PQ pool.By monitoring the nonphotochemical quenching and state transition processes,it suggested that lack of SpkI also caused negative effects on the photoprotection mechanisms of Synechocystis.Moreover,SpkI was involved in regulating the photoinhibition effects of PSⅡ upon high salt combined with high light conditions.To further explore the possible substrates of SpkI and PphA,a quantitative phosphoproteomic study was performed in WT,Δspkl and ΔpphA.It revealed that lack of SpkI or PphA altered the phosphorylation status of key proteins participated in photosynthetic-related processes,and SpkI and PphA may work as counterpart on regulation of the phosphorylation states of some crucial proteins in Synechocystis.In this work,we clarified for the first time the physiological significance of Ser/Thr protein kinase SpkI upon high salt condition in Synechocystis.The first quantitative phosphoproteomic data of ΔspkI and ΔpphA enriched our knowledge of protein phosphorylation regulating networks in Synechocystis.Our findings would shed light on the molecular basis of protein phosphorylation involved in the regulation of photosynthetic-related processes,and provide novel insights in high salt acclimation mechanisms in cyanobacteria.