Protein Phosphorylation And Lys-acetylation Analyses And Molecular Physiology Of Mangrove Kandelia Candel In Response To Flooding

Flooding is one of the major environmental stresses.Global warming is the cause of increasingly frequent flooding in many lands in the world,under which crop plants experience energy deficiency or even death resulting in large economic losses.Kandelia candel(L.)Druce,a kind of woody mangrove,experiences daily flooding cycles in its natural habitat.To explore the molecular mechanisms underlining the physiological adaptation of K.candel to flooding,K.candel seedlings without flooding(namely Control group)and with flooding for 6 h(namely Flooding group)were selected as materials.Regulations of protein phosphorylation and Lys-acetylation in flooding responses were first investigated by using a large-scale quantitative phosphoproteome and lysine acetylome methods,respectively.The qPCR analyses and physiological observations relevant to flooding responses were together carried out to reinforce that K.candel is a species adapted to flooding.In addition,cloning and expression analyses of RCA gene from the leaves of K.candel were performed to reveal its role in response to flooding conditions.(1)The results of phosphoproteome in K.candel leaves showed that:i)A total of 2603 non-redundant phosphorylation sites and 2141 unique phosphopeptides were identified from 1516 unique phosphoproteins in K.candel leaves;ii)Three new motifs[GSP],[G××SP],and[RS×S]out of all the 10 motifs were discovered;iii)The phosphorylated level of 96 proteins were found to be significantly changed(more than 1.5-fold,P?0.05),among which 75%differentially phosphorylated proteins in Flooding group were up-regulated as compared with Control;iv)Based on GO,KEGG and protein function analyses,the 96 differentially phosphorylated proteins were mainly involved in pyruvate metabolism and energy production,as well as protein synthesis and phosphorylation.Furthermore,the consistent results at the phosphoproteomic and transcriptional levels suggested to demonstrate that regulations of the pyruvate metabolism and energy production pathway including respiration and photosynthesis could contribute to K.candel's flooding adaptation.(2)The results of lysine acetylome in K.candel leaves showed that:i)A total of 1041 lysine acetylation(LysAc)sites,1021 Lys-acetylated peptides and 617 Lys-acetylated proteins were identified;ii)Six conserved sequence motifs of the LysAc sites,including a new motif[KxxxxK],were detected,in which the residues in the upstream and downstream of acetylated K were enriched for E,R and K;iii)The LysAc level of 260 Lys-acetylated proteins were found to be differentially changed more than 1.5-fold in response to flooding,among which 60%differentially Lys-acetylated proteins in Flooding group were up-regulated as compared with Control;iv)Based on GO,KEGG and protein function analyses,66 proteins(more than 1.5-fold,P<0.05)were enriched in carbohydrate and energy metabolism,photosynthesis,and stress response and defense pathways in response to flooding.Additionally,coupled with transcriptional analyses,the findings indicated that the carbohydrate metabolism and photosynthesis in flooding-responsive K.candel leaves were found to be regulated together by reversible protein LysAc and transcription.(3)The physiological observations in K.candel leaves showed that:i)The activities of SOD and POD were significantly increased,and the antioxidant enzyme activities and antioxidant contents in AsA-GSH cycle both were obviously up-regulated in response to flooding,which accordingly enhanced the antioxidant ability;ii)Compared with the control seedlings,the ATP levels did not change significantly,the pyruvate level was distinctly increased,and the NAD+and acetyl-CoA contents were significantly decreased in flooded leaves;iii)The activities of PDH,CS,ICDH,OGDH,MDH and PEPC in TCA cycle remained unchanged or decreased dramatically in response to flooding;iv)The activities of PFK,GAPDH,PK,PDC and ADH were increased significantly in flooding-responsive K.candel seedlings;v)The activities of two key enzymes G6PDH and 6PGDH in pentose phosphate pathway were up-regulated notably in the Flooding group;vi)The Pn in flooded K.candel leaves was significantly decreased to 79.3%of that in Control,and the activities of both Rubisco and RCA were drastically decreased in response to flooding,nevertheless the chlorophyll contents and PSII structure were not affected by flooding;vii)The sucrose content and the activities of SPS and SuSy were all increased significantly in response to flooding.These results strongly suggested that K.candel leaves could eliminate excessive ROS and consequently reduce the ROS damage by increasing the activity of POD and SOD and enhancing the ability of AsA-GSH cycle,meanwhile it could acquire stable energy supply during daily flooding by channeling pyruvate to anaerobic respiration,increasing PPP activities,maintaining a relatively high level of photosynthesis and accumulating sucrose contents.Most of key enzymes in these metabolic pathways were phosphorylated or lysine acetylated in flooding-responsive K.candel leaves.(4)Through the cloning and expression analyses of RCA gene,the results showed that:i)KcRCA,namely RCA gene from K.candel leaves,had two types with long chain and short chain,respectively defined as KcRCAL(GenBank accession number:MG492021)and KcRCAs(GenBank accession number:MG492022).The cDNA sequence of KcRCAL contained an open reading frame(ORF)of 1461 bp encoding 440 amino acids which belonged to the ? subunit(small subunit)of RCA as the theoretical molecular mass of the maturation KcRCAL protein was 42.49 kDa,while The cDNA sequence of KcRCAs contained ORF of 1461 bp encoding 474 amino acids which belonged to the a subunit(large subunit)of RCA as the theoretical molecular mass of the maturation KcRCAs protein was 46.10 kDa;ii)The proteins KcRCAL and KcRCAs both contained phosphorylation sites and LysAc sites;iii)The phylogenetic analysis based on the amino acid sequences showed that KcRCA had the closest genetic relationship with the RCA from chickpea(Cicer arietinum)and durian(Durio zibethinus);iv)KcRCAL and KcRCAs overexpressing transgenic Arabidopsis thaliana plants increased antioxidant abilities and the increased activities of RCA and Rubisco,which contributed to enhancing photosynthesis under waterlogging stresses;v)The expression levels of KcRCAL and KcRCAs were up-regulated in flooding-responsive K.candel leaves,which contributed to resisting flooding stresses.Taken together,K.candel seedlings stabilize energy supply through maintaining sufficient photosynthesis activities,achieving effective anaerobic respiration and increasing pentose phosphate pathway flux.Reversible protein phosphorylation and LysAc are likely to be important post-translational regulatory mechanisms for enzyme activity as well as for photosynthesis,anaerobic respiration and PPP in K.candel to adapt to flooding.Increased regulations of KcRCAL and KcRCAs play vital roles in response to flooding stresses in K.candel.KcRCA overexpressing transgenic Arabidopsis thaliana plants defense waterlogging stresses through increasing antioxitant aiblity and regulating photosynthesis.This study will provide a new test basis for further improvement on flooding-stressed mechanism in plants,and also provide novel insights into breeding flooding-tolerant species.