The Mechanism Of Chloroplast AtRabF1 In Tolerance To Salt Stress And Senescence And Phosphatidylcholine Uptaken In Vitro

Chloroplasts are important for photosynthesis in plants.They use solar energy to assimilate CO₂ and H20,and finally produce 02 and carbohydrate.Meanwhile,chloroplast is also an important sensor for stress signaling perception in plants.They can synthesize amino acids,fatty acids,sulfur and nitrogen metabolism which would be the precursor for some signal molecue to reponse for stresses.It is meaningful to do the research on chloroplast proteins and lipids for improving photosynthesis of crops,enhancing resistance to abiotic stress and increasing the amount of products of crops.Rab?Ras-like protein in brain?proteins are a kind of molecule switch and playing important roles during regulating vesicle formation,transport,anchoring and fusion between vesicles and acceptor membrane system,and meanwhile,they are very vital in substance transporting,developing,biotic and abiotic stress.AtRabA5e was involved in cold and oxidative stress and founded in the stroma and thylakoid in chloroplasts.The localization and functional characterization of Arabidopsis AtRabF1 was studied in this paper.The genetic interaction between AtRabFl and AtRabA5e was studied by atrabF1-1/atrabA5e double mutant.Chloroplasts can not synthesize PC?Phosphatidylcholine?by itself and the PC in chloroplasts is transported from cytosol to chloroplast membrane system.The transport model of PC to chloroplasts was studied in vitro in this paper.Main results were below.1.By fluorescence co-localization and Western blot,AtRabFl was localized on the chloroplasts' envelope and thylakoid.When AtRabF1 was mutated into constitutively active?CA?mutation,the CA-AtRabF1 had similar localization as wild AtRabF1,which localized on the envelope and inside of chloroplast.However,when AtRabF1 was mutated into dominant negative?DN?mutation,it was found that the DN-AtRabF1 was mainly assembled on the surface of chloroplast and no fluorescence was found inside of chloroplast.The localization of WOTPRabF1?trunctated AtRabFl without predicted transit peptide?was the same with DN-AtRabF1.All these indicated that the active condition was indispensable for AtRabFl entering into chloroplast successfully.Myristoylation and palmitoylation at N-terminus are indispensable for AtRabF1 protein anchoring on the membrane.The trunctated AtRabF1 pretein was disturbed to anchoring and entering to chloroplast.2.Comparing Col-0 and AtRabFl knockout mutants,no visible phenotype was found.Over-expressing AtRabFl,CA-AtRabF1 and DN-AtRabFl in atrabF1-1 knockout mutant and comparing with WT and atrabF1-1,no visible phenotype difference was found either.Four days old seedlings were transferred to MS plates containing 100 mM NaCl and stressed for another 6 days.It was found that all the overexpressing complementary lines?including AtRabF1 OE lines,CA-AtRabF1 OE lines and DN-AtRabF1 OE lines?had significant longer root length than WT and knockout mutants,and no significant difference was found between WT and knockout mutants.However,the chlorophyll content of all the overexpressing complementary lines was significant lower than WT after 6 days salt stress.In the same time,the proline content of all the overexpressing complementary lines was significant lower than WT after salt stress.Four days old seedlings were transferred to MS plates containing 50?M ABA for growing another 6 days.The results showed that all the overexpressing complementary lines had significant longer root length than WT and knockout mutants.Comparing the proline content after 6 days ABA treatment,it was also found that all the overexpressing complementary lines had significant lower proline than WT.All the results indicated that overexpressing AtRabFl,CA-AtRabF1 and DN-AtRabF1 can significantly enhance the ability of resistance of transgenic plants to salt stress.Proline content was as damage index during stress in salt stress since stress sensitive WT and knockout mutants had higher content.It is speculated that the overexpressing complementary lines were in ABA dependent pathway during resistant to salt stress.3.The fouth leaves from WT and AtRabFl knockout mutants were detached and treated with 2 days and 4 days dark-induced-senescence respectively.No visible phenotype,chlorophyll content and leaf conductivity difference was found between WT and AtRabFl knockout mutants.However,the expression level of SAG12?Senescence Assiciated Gene?and SEN1?Senescence?in knockout mutants was much higher than in WT,and the expression level of CAB1?Chlorophyll A/B binding protein?and RBCS1A?Ribulose Bisphosphate Carboxylase Small Chain 1A?were much lower than in WT,which indicated that knocking out AtRabFl can accelerate senescence associated genes' expression.Overexpressing AtRabF1,CA-AtRabF1 and DN-AtRabF1 complementary lines had higher resistance to dark induced senescence than WT and knockout mutants,showing greener leaves,higher chlorophyll content and lower leaf conductivity.4.There was no visible phenotype difference by comparing WT,atrabF1-1 and atrabA5e mutant and double mutant under normal growth condition.atrabA5e and double mutant atrabF1-1/atrabA5e had significant longer root length than WT and atrabF1-1 after 100 mM NaCl treatment,however,both of them showed lower chlorophyll and proline content than WT like AtRabFl overexpressing lines.After treated with 50?M ABA,both atrabA5e and atrabF1-1/atrabA5e had significant longer root length than WT and atrabF1-1,meanwhile containing lower proline content.After dark induced senescence,atrabA5e and atrabF1-1/atrabA5e exhibited stronger resistant to senescence than WT and atrabF1-1,showing greener leaves,higher chlorophyll content and low conductivity.Hence,AtRabA5e was working as negative factor during salt stress and dark induced senescence.5.There was no significant difference of PS?parameters between WT and AtRabFl knockout mutants both under normal growing condition and 200 mM NaCl stressed for 7 d.However,under normal growing condition and 200 mM NaCl treatment for 7 d respectively,overexpressing AtRabFl,CA-AtRabF1 and DN-AtRabFl affect ABS/RC,ETo/RC,DIo/RC,PIABS and PItotai on PS?,which indicated that overexpressing AtRabF1,CA-AtRabF1 and DN-AtRabF1 have effect on PS?in some ways.6.The total phospholipids of pea were extracted from pea seedlings labeled by H₃³³PO₄ and were used to make liposomes.The transport of total phospholipids from liposomes to chloroplasts was time dependent in vitro.Arabidopsis PC was extracted from Arabidopsis leaves labeled by 14C-sodium acetate.The liposomes made from 14C labeled Arabidopsis PC and commercial 14C-DPPC taken up by isolated pea chloroplasts in vitro was time and tempreture dependent respectively.After the digestion of proteins on the outside of chloroplast outer envelope by thermolysin,the rate of PC taken up by treated chloroplasts decreased a lot,which indicated that proteins on the chloroplast outer envelope can assit chloroplasts to take up PC.When ATP or GTP was added to the recaciton during PC transport in vitro,there was no increase of uptake rate,which indicated that energy was dispensable during chloroplasts taken up PC in vitro.However,when the size of liposomes made from 14C-DPPC was increased,it was found that the uptake rate of PC was increasing with the size of liposomes.It was speculated that during the transport of PC from the liposomes?or PC from ER?to chloroplasts,the membrane contact site?MCS?was formed between different membrane system and the MCS can be a kind of hemifusion,meanwhile the proteins on the chloroplast outer envelope at the MCS were also very vital for chloroplasts taking up PC.In summary,we founded that the chloroplast protein AtRabF1 plays a vital role in salt and dark induced senescence strees,and understanded the transport model of phophatidylcholine to chloroplast.It has very important theoretical meaning for elucidating the function and mechanism of chloroplasts' proteins and lipids in abiotic stress by our research.It is also very meaningful for crops genetic improvement and breeding in abiotic stress in future.