The Functions And Their Mechanisms Of Plant Cysteine Oxidases And Hydrogen Sulfide In Response To Low Oxygen Stress In Arabidopsis

In animals and plants,oxygen-dependent branch of N-end rule pathway mediates protein N-terminal cysteine oxidation,which determines stability of specific proteins.Recently,it has been found that plant cysteine oxidases?PCOs?use O2 as a substrate to oxidize the N-terminal cysteine of ethylene response transcription factors VII?ERFVII?,thereby controlling their stability and lifetime.Hence,the ERFVII proteins are stable under low oxygen and activate molecular hypoxic response,while the anaerobic response is suppressed in air.The PCOs is comprised of five members in Arabidopsis.PCO1 and PCO2 were shown to act as part of an O2 sensing mechanism,while the function of other PCO members,including PCO3,PCO4 and PCO5,still remains unclear.In addition,the gas transmitter hydrogen sulfide?H₂S?is shown to act as an O2 sensor in animals,which is involved in hypoxia-induced disease regulation.However the role of H₂S in plant hypoxia responsive signal pathway remains unclear.In order to explore the characterization of the PCOs and H₂S in oxygen sensing mechanism and molecular hypoxia response,we performed a series of experimental analyses,including morphological comparison,physiological and biochemical detection,bioinformatics analysis,quantitative real-time reverse transcription polymerase chain reaction?q RT-PCR?and genetic engineering technology,in Arabidopsis wild type?Col-0?and PCO-associated T-DNA insertion mutants.The main results are as follows:1.The statistical analyses of seed germination percent and rate of WT and PCO-related mutants presented that pco1pco2,pco4pco5 and pco1pco2pco4pco5 showed delayed germination compared with WT and PCO single mutants.And germination percent of pco1pco2pco4pco5 was almost 45%,only half of WT.2.Through analyzing the phenotypic characters,such as root length,rosette leaf number and leaf area,of WT and PCO-related mutants grown under normal conditions,it was found that the primary root length of pco1pco2?pco4pco5 and pco1pco2pco4pco5 were shorter than WT and PCO single mutants.The leaves of them were smaller,and the rosette leaf number decreased significantly.Especially for pco1pco2pco4pco5,the growth and development of it was much slower and did not produce seeds.The leaf number of pco1pco2pco4pco5 was much less 50% than that of wild type.The leaf area of pco1pco2pco4pco5 was only one third of wild type.Meanwhile,there was not obvious phenotypic differences between WT and PCO single mutant,indicating that PCOs may act redundantly in plant cells.3.Combining phylogenetic analysis of PCO family across different plant species with amino acid sequence and transcriptional correlation of these proteins,PCOs could be subdivided into two clades,clade I,including PCO1 and PCO2,that are upregulated upon hypoxia,and clade II whose expression is constitutive and unaffected by low oxygen,including PCO3,PCO4 and PCO5.4.The subcellular localization of PCO3,PCO4 and PCO5 using AGROBEST transient transformation showed that PCO4 and PCO5 were both localized in nucleus and cytosol,similar to PCO1 and PCO2,while the PCO3 was only localized in cytosol.5.Fusion of the promoter of PCO3 and PCO4 with a GUS:GFP reporter confirmed that PCO3 and PCO4 expressed constitutively in Arabidopsis.Additionally,hypoxic stress could induce expression of PCO3 in plant root elongation zone,and PCO4 expression was mainly induced in leaves and root primodium upon oxygen deprivation.6.Our q RT-PCR results revealed that PCO-related knock out mutants grown under air conditions could lead to constitutive induction of anaerobic marker genes,which was consistent with gene expression in prt6 and ate1ate2.This demonstrated that clade II in Arabidopsis are also involved in the hypoxic response,indicating that N-end rule mediated oxygen sensing in plants is regulated by both hypoxia-inducible PCOs clade I and non-inducible clade II.7.Evans blue and DAB staining results displayed that only few part of leaves pretreated with H₂S were stained with blue after 3 d of submergence treatment,and staining of DAB were lighter than those seedlings pretreated without H₂S.Additionally,exogenous H₂S pretreatment had no effect on SOD?superoxide dismutase?activity during submergence,while the content of GSH?glutathione?was obviously increased after 36 h of submergence treatment pretreated with H₂S.Moreover,q RT-PCR analysis revealed that the expression level of programmed cell death related marker gene PR1 was significantly downregulated upon air and submergence conditions pretreated with H₂S,illustrating that exogenous H₂S pretreatment decreased the H2O2 accumulation and alleviated the cell death degree of Arabidopsis seedlings upon submergence treatment.8.The observation of H₂S specific fluorescence probe showed exogenous H₂S pretreatment promoted the production of intracellular H₂S in Arabidopsis root tips,and confirmed the induction of H₂S by low oxygen stress.Furthermore,q RT-PCR test indicated that expression levels of H₂S-related metabolic marker genes,namely DES1?L-cysteine desulfhydrase 1?,CYS-C1?cyanoalanine synthase-C1?and OASA1?O-acetylserine?thiol?lyase?OAS-TL?isoform A1?,were significantly enhanced after pretreatment with H₂S,which is beneficial to maintain the optimal endogenous H₂S levels in Arabidopsis under both air and hypoxia conditions.9.During submergence treatment,exogenous H₂S pretreatment affected the transcriptional expression levels of ER stress-induced unfolded protein response?UPR?marker genes,including ZIP17?basic leucine zipper 17?,ZIP28?basic leucine zipper 28?,BIP1?binding protein 1?,BIP3?binding protein 3?,TIN1?tunicamycin induced 1?,PDI6?protein disulphide isomerase 6?,ERO1?ER oxidoreductase 1?,CNX1?calnexin 1?and CRT1?calreticulin 1?,implying that H₂S could improve submergence resistant ability of Arabidopsis via influencing the proper protein folding process.In conclusion,the entire PCO family in Arabidopsis are all involved in regulating oxygen sensing mechanism via oxygen-dependent branch of the N-end rule pathway,thereby controlling the anaerobic response in plant.For another thing,H₂S could regulate Arabidopsis adaptation for submergence-induced low oxygen stress through influencing the correct folding of misfolded protein.In this study,it is promising to broaden the future research idea in unraveling the direct or indirect effect of H₂S on oxygen sensing pathway in plant via combining N-end rule mechanism with H₂S together,which is beneficial to provide sufficient theoretical basis and put it into practice in crop improvement.