Molecular Mechanism Of Hydrogen Sulfide As Gasotransmitter Promotes Flowering In Chinese Cabbage

Hydrogen sulfide(H2S)is the third gasotransmitter found after nitric oxide and carbon monoxide.In plants,it was found that the physiological concentration of H2S could promote the germination of seeds,accelerate the formation of root morphology,regulate the stomatal movement,enhance the photosynthesis of leaves and improve the resistance to various abiotic stress in plants.H2S helps plants to resist to drought stress,salt stress,high temperature stress,hypoxia stress,heavy metal stress and so on.Flowering is a vital important transformation process from vegetative growth to reproductive growth in higher plants.The study of flowering regulation mechanism has important theoretical significance and application value.In the model plant Arabidopsis thaliana,many pathways involved in florescence regulation have been found,including vernalization pathway,photoperiod pathway,gibberellin pathway and autonomous pathway.Chinese cabbage(Bassica rapa L ssp.pekinensis)is a cruciferous brassica vegetable,and is one of the most important vegetable crops in northern China.Its regulation of flowering is also a hot research topic.There is a linear correspondence between the genes in Chinese cabbage and that in Arabidopsis.However,Brassica rapa represents a very close relative,with its triplicated genome due to an ancient genome duplication event.Therefore,one gene in Arabidopsis can often find multiple copies in Chinese cabbage.The intriguing question is whether these different homologys in Chinese cabbage are only multiple copies with functional redundancy,and play similar roles compared with Arabidopsis? Or they are assigned to different roles? It still remains an open question regarding the functional conservation and diversification of duplicated homologys in determining flowering time in Chinese cabbage.In our previous work,it was found that H2S had a significant effect on promoting Arabidopsis and chinese cabbage flowering.Here,the molecular mechanism of H2S promoting flowering was studied with Chinese cabbage as experimental material.The experimental results are as follows.1.Exogenous H2S with concentration of 100 μmol·L-1 promoted the flowering of Chinese cabbage significantly.Compared with the control group,it is more effective when plants vernalized for only 8-9 days.The use of the inhibitor of H2S synthesis(HA)shows the opposite effect.The results showed that exogenous application of H2S could promote flowering by compensating for insufficient vernalization to some extent.In long day condition,application of exogenous H2S promoted the flowering,and HA inhibited the flowering in Chinese cabbage.However,In short day condition,no flowering was observed in exogenous application of H2S and HA,even in CK groups.The relationship between H2S and photoperiod pathway can not yet be determined completely.GA treatment promoted the flowering of Chinese cabbage significantly,while the application of HA did not weaken the effect of GA.These data indicated that H2S was not the downstream signal of GA pathway.However,whether H2S signal affects GA synthesis still remains unknown.2.Bra FLCs has four homologys in Chinese cabbage,Bra FLC 1,Bra FLC 2,Bra FLC 3 and Bra FLC 4.Bra FLC 1-3 transcription factor contains typical MADS-box and k-box,but in Bra FLC 4,K-Box domain was missing and only MADS-box was present.Bra SOCI,as a downstream gene of Bra FLCs,has five copies of homologys in Chinese cabbage.The promoters of these genes contain eight CC(A/T)6AG(CAr G-box)binding sites for Bra FLCs.We analysised the binding effect of 4 Bra FLCs proteins with 8 Bra SOCIs promoter probes.The results showed that Bra FLC 1 bound to the first CAr G-box in SOCI 1 promoter,the second CAr G-box in SOCI 3 promoter,the CAr G-box in SOCI 4 and SOCI 5 promoter;Bra FLC 2 bound to the CAr G-box in SOCI 4 and SOCI 5 promoter;Bra FLC 3 bound to the first CAr G-box in SOCI 2 promoter,and the CAr G-box in SOCI 4 and SOCI 5 promoter;and Bra FLC 5 bound to the CAr G-box in SOCI 5 promoter only.Different Bra FLC proteins bound to different CAr G-box in the promoter of Bra SOCI binding sites,showed different binding patterns.It strongly implied the functional diversification of Bra FLCs and Bra SOCIs homologys existed in Chinese cabbage.3.It has been reported and believed that H2S can regulate the proteins function by cystein S-sulfhydration modification.Our experiments In vitro showed that only Bra FLC 1 and Bra FLC 3 of the Bra FLCs could undergo S-sulfhydration modification by H2S.Correspondingly,the binding ability of modified Bra FLC 1 and Bra FLC 3 proteins to most Bra SOCIs promoter probes weakened or even disappeared,which showed clearly the possible ways of H2S signal in regulating the flowering time.In conclusion,it could be speculated that the possible way of promoting flowering by H2S in Chinese Cabbage is that H2S in physiological concentration can modify part of the members of Bra FLCs transcription factors by S-sulfhydration.The binding ability of modified Bra FLCs to downstream target promoter was weakened or blocked completely.In view of the fact that Bra FLCs are the key negative regulatory factor in the flowering process in response to vernalization.Therefore,the effect of H2S signal on flowering time was to promote the transformation from vegetative growth to reproductive growth ultimately.In short,H2S signaling promoted plant flowering by weakening or eliminating the binding ability of Bra FLCs to downstream promoters through S-sulfhydration modification.The discovery of this phenomenon revealed the diversity of flowering regulation modes and functional diversification of Bra FLCs homologys in Chinese cabbage.On the other hand,It provided new clues for the study on physiological functions of H2S.