The Function Of Putrescine Regulation And PGR5-dependent Cyclic Electron Flow In Salt Tolerance In Cucumber

Cucumber(Cucumis sativus L.)is one of the most widely cultivated protected vegetables in China,but various stresses such as extreme temperature and salinity have seriously affected the yield and quality of cucumber,so it is instructive to explore the stress response mechanism of cucumber to relieve stress damage and improve its yield and quality.The cyclic electron flow(CEF)around photosystem I(PSI)is one of the most important photoprotection mechanisms in photosynthesis.There are two CEF pathways in plants:the NADPH dehydrogenase(NDH)-dependent and the proton gradient regulation 5(PGR5)dependent CEF,of which,PGR5-CEF plays a major role in angiosperms,however,its specific mechanism is still very vague,and no detailed analysis of the PGR5 protein itself,besides,it is unclear that which complexes it binds to and how it works.At present,these researches about PGR5-CEF are mainly focused on model materials such as cyanobacteria and Arabidopsis,but little research has been done on other plants.In this study,cucumber was used as test material.Firstly,we studied the important mechanism of how putrescine(Put)coordinates PGR5-CEF to alleviate the damage caused by salt stress in photosynthesis.Secondly,we used the cucumber genome database to analyze PGR5 members in cucumber and made a comprehensive analysis of cucumber PGR5 by bioinformatics.Thirdly,we screened the interaction proteins of PGR5a in cucumber by the yeast two-hybrid system and analyzed the protein complexes it binding to.Finally,we revealed the role of cucumber PGR5-CEF in response to salt stress by genetic transformation technology.The main results obtained are as follows:The two photosystems of cucumber seedlings suffered severe but reversible photoinhibition under NaCl stress.Salt stress compelled photosynthetic apparatus to induce high level of CEF for self-protection,although this protection mechanism can ensure the survival under salt stress,the growth of cucumber seedlings was still significantly inhibited.After spraying of Put,the CEF level of cucumber seedlings was increased about 30%.Saltstressed plants which treated with Put showed higher levels of CEF and 40%more ATP accumulation than those only treated with salt stress,thus providing appropriate ATP/NADPH ratio for normal growth.Furthermore,the CEF-induced increase of transmembrane proton gradient(△pH)is reduced by Put,and accompanied by a decrease in pH-mediated non-photochemical quenching(qE),thus,more excitation energy is utilized by photochemical quenching.Under the coordination of Put and CEF,the NADP+/NADPH ratio in the salt-stressed leaves increases significantly,indicating that the over-reduction pressure at the PSI receptor site was released.Overall,our results proved that exogenous Put creates superior conditions for the induction of CEF,and higher levels of CEF alleviates the photoinhibition of the photosystems under salt stress,protecting the photosynthetic apparatus and making the plants more salt-tolerant.Two members of CsPGR5,named CsPGR5a and CsPGR5b,were identified in the cucumber genome database.They were located on chromosomes 2 and 3,respectively.The full length of CDS were both 384 bp,encoding 127 amino acids.We aligned the amino acid sequence of PGR5 in multiple species by using the amino acid sequence of AtPGR5 as a probe,and found that there were commonly two PGR5s in some algae and higher plants,and the mature part of PGR5 amino acid sequence was highly conserved among various species.The results of gene expression analysis showed that CsPGR5a and CsPGR5b had similar expression patterns in different tissues of cucumber and response to hormone induction and different stress conditions,and the expression level of CsPGR5a is significantly higher than that of CsPGR5b.These results indicated that PGR5 was a highly conserved protein that can participate in various environmental responses,and CsPGR5a played a dominant role in PGR5-CEF in cucumber.In order to further explore the role of CsPGR5a on thylakoid membranes,we used the yeast two-hybrid system to screen cucumber cDNA libraries,and candidate proteins that could interact with CsPGR5a were initially obtained.It was further verified that PGR5a interacted with Lhcb3,PetC,and PsaH in cucumber by using the yeast two-hybrid,twomolecule fluorescence complementary(BiFC),blue-native PAGE(BN-PAGE),and coimmunoprecipitation(CoIP)experiments.The interaction between PGR5a and the lightharvesting protein subunit Lhcb3 of PSII suggested that the binding of PGR5a to PSI may be related to state transition.In addition,PGR5a interacted with PetC in the cyt b6f complex,indicating that PGR5a may have similar functions as PetC to regulate the proton gradient across thylakoid membranes.In order to verify the relationship between PGR5 and state transition,we induced state transition of thylakoids by light and chemical methods,and found that when in state 1,most PGR5 bound to the cyt b6f complex,and regulated the transport of electron and proton;When state transited from state 1 to state 2,part of PGR5 dissociated from cyt b6f and bound to PSI,thereby regulating the electron flow at the PSI to maintain the stability of the photosystems.In addition,after over-expression of CsPGR5a in tobacco,we found that the high-expression lines grew smaller,had thicker and darker leaves;after treated with 100 mM NaCl,the salt tolerance of the CsPGR5a-overexpression lines was significantly superior to that of the wild type,and the ability of salt tolerance was enhanced with the increasing of expression level.The high-expression lines could maintain high photosynthetic properties of PSI under the compound stresses of fluctuating light and salt stress,indicating that PGR5-mediated CEF played an extremely important role in improving salt tolerance of plants.