| Isoflavones,an important group of flavonoids,belong to a family of polyphenols and are synthesized predominantly in legumes.Enrichment of isoflavones has been investigated in recent decades as a result of their potential for health-related benefits to humans.Isoflavone accumulation is regulated by environmental stimuli,especially lights.UV-B radiation has been considered to be potential way for improving the expected secondary metabolite levels.The ultra-violet(UV)-absorbing characteristics of flavonoids have long been considered to be the reason why flavonoids could protect plants from UV damage.However,UV-B stress,as an external stressful signal,is not directly involved in the secondary metabolism of plants,instead of activating its corresponding effectors.The objectives of this study are to systematically explore mechanisms of UV-B radiation-induced isoflavone accumulation in soybean sprouts from the perspective of intracellular signal transduction.The main results were as follows:1.The induction of isoflavone accumulation by abscisic acid(ABA)in soybean sprouts under UV-B radiation was investigated.UV-B radiation activated 9-cis-epoxycarotenoid dioxygenase(NCED),leading to enhancement of ABA synthesis.Both of UV-B radiation and ABA treatments stimulated endogenous ABA accumulation.The highest ABA level under UV-B radiation and ABA treatments was observed on day 4,which was 1.9 and 2.4 times higher than control,respectively.UV-B radiation and ABA treatments also induced isoflavone synthesis.The highest isoflavone level under UV-B radiation and ABA treatments was observed on day 4,which was 1.5 and 1.4 times higher than control,respectively.The supply of UV-B radiation and exogenous ABA together caused a greater increase in endogenous ABA and isoflavone content.These data indicated that isoflavone accumulation in soybean sprouts was activated by UV-B radiation possibly with ABA acting downstream in the signaling pathway.2.The induction of isoflavone accumulation by nitric oxide(NO)in soybean sprouts under UV-B radiation was investigated.NO level in soybean sprouts treated with UV-B radiation was much higher than control.The highest NO level was observed on day 2 after UV-B treatment,which was 5.8 times higher than control.This resulted from the enhancement of nitric oxide synthase(NOS)activity.2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO)(NO scavenger)inhibited UV-B-induced NO production.The inhibition was reversed through application of exogenous NO donor sodium nitroprusside(SNP).This suggested that NO production was truly induced by UV-B radiation.What’s more,cPTIO inhibited UV-B radiation-induced isoflavone content,chalcone synthase(CHS)activity,isoflavone synthase(IFS)activity and CHS expression and IFS expression by 32%,42%,26%,23%and 43%in 4-day-old soybean sprouts respectively.SNP could reverse the inhibition caused by cPTIO.Thus,NO was an essential signaling molecule mediating UV-B radiation-induced isoflavone accumulation in soybean sprouts.3.The interaction between ABA and NO in soybean sprouts under UV-B radiation was investigated.After UV-B radiation and ABA treatments,the NO level was 5.1 and 6.3 times higher than control and NOS activity was 2.7 and 2.8 times higher than control in 4-day-old soybean sprouts respectively.The supply of UV-B radiation and exogenous ABA together caused a greater increase in endogenous NO content and NOS activity.These data indicated that NO production in soybean sprouts was activated by UV-B radiation possibly with ABA acting downstream in the signaling pathway.cPTIO inhibited UV-B radiation-induced ABA content,activity and gene expression of NCED by 26%,28%and 41%in 4-day-old soybean sprouts respectively.SNP could reverse the inhibition caused by cPTIO.SNP treatment alone could also induce ABA synthesis.In addition,the protein expression of two core components of ABA signaling:type 2C protein phosphatase(PP2C)and SNF1-related protein kinase(SnRK)was regulated by NO.Thus,NO is an essential signaling molecule mediating UV-B-induced ABA accumulation in soybean sprouts.4.The mediation by guanosine 3’,5’-cyclic phosphate/protein kinase G(cGMP/PKG),a downstream signal of ABA and NO,of UV-B radiation-induced isoflavone accumulation in soybean sprouts was investigated.cGMP level,activity and protein expression of guanylyl cyclase(GC)in the soybean sprouts treated with UV-B radiation were much higher than control.The highest cGMP level,activity and protein expression of GC were observed on day 4 after UV-B treatment,which were 2.4,2.3 and 1.2 times higher than control.6-Anilino-5,8-quinolinequinone(LY83583)(GC inhibitor)inhibited UV-B radiation-induced cGMP level,activity and protein expression of GC by 52%,27%and 16%in 4-day-old soybean sprouts respectively.The inhibition could be reversed by ABA,SNP and 8-cGMP.ABA,SNP and 8-Br-cGMP alone could also up regulate cGMP level.ABA and SNP could enhance activity and protein expression of GC.Therefore,ABA and NO activated GC,leading to cGMP production under UV-B radiation.What’s more,LY83583 and KT5823(PKG inhibitor)inhibited UV-B radiation-induced isoflavone content by 37%and 24%,CHS activity by 40%and 39%,IFS activity by 24%and 25%,gene expression of CHS by 18%and 29%,gene expression of IFS by 41%and 32%,protein expression of CHS by 56%and 59%,protein expression of IFS by 86%and 78%in 4-day-old soybean sprouts respectively.ABA,SNP and 8-cGMP could reverse the inhibition.ABA,SNP and 8-cGMP alone could also up regulate isoflavone content,activity and expression of CHS and IFS.Thus,GC/cGMP acted as a downstream signal of ABA and NO to induce isoflavone accumulation via a PKG-dependent pathway.5.The mediation by inositol 1,4,5-trisphosphate(IP3)of isoflavone accumulation induced by ABA-and NO-triggered cGMP in soybean sprouts under UV-B radiation was investigated.After UV-B treatment,IP3 content and activity,gene as well as protein expression of phosphoinositide-specific phospholipase C(PI-PLC)in 4-day-old soybean sprouts were 2.5,2.5,3.3 and 2.1 times higher than control.LY83583 and KT5823 inhibited UV-B radiation-induced IP3 content and activity,gene as well as protein expression of PI-PLC in 4-day-old soybean sprouts.ABA,SNP and 8-cGMP could reverse the inhibition.ABA,SNP and 8-cGMP alone could also up regulate IP3 content and activity,gene as well as protein expression of PI-PLC.Therefore,ABA-and NO-triggered cGMP activate PI-PLC,leading to IP3 accumulation under UV-B radiation.What’s more,neomycin(PI-PLC inhibitor)inhibited UV-B radiation-induced isoflavone content by 32%,activity of CHS and IFS by 46%and 53%,gene expression of CHS and IFS by 55%and 36%,protein expression of CHS and IFS by 67%and 69%in 4-day-old soybean sprouts,respectively.ABA,SNP and 8-cGMP could reverse the inhibition.Thus,IP3 as the second biomessengers of cGMP/PKG,mediated ABA-and NO-triggered isoflavone production under UV-B radiation.6.The mediation by cADP-ribose(cADPR)of isoflavone accumulation induced by ABA and NO-triggered cGMP in soybean sprouts under UV-B radiation was investigated.After UV-B treatment,cADPR content and activity as well as protein expression of ADP-ribosyl cyclase(ADPRC)in 4-day-old soybean sprouts were 2.9,1.8 and 1.8 times higher than control.LY83583 and KT5823 inhibited UV-B radiation-induced cADPR content and activity as well as protein expression of ADPRC in 4-day-old soybean sprouts respectively.ABA,SNP and 8-cGMP could reverse the inhibition.ABA,SNP and 8-cGMP alone could also up regulate cADPR content and activity as well as protein expression of ADPRC.Therefore,ABA-and NO-triggered cGMP activated ADPRC,leading to cADPR accumulation under UV-B radiation.What’s more,nicotinamide(cADPR inhibitor)inhibited UV-B radiation-induced isoflavone content by 41%,activity of CHS and IFS by 32%and 44%,gene expression of CHS and IFS by 22%and 23%,protein expression of CHS and IFS by 36%and 38%in 4-day-old soybean sprouts,respectively.ABA,SNP and 8-cGMP could reverse the inhibition.Thus.cADPR,as the second biomessengers of cGMP/PKG,mediated isoflavone production under UV-B radiation.7.The mediation by mitogen-activated protein kinase(MAPK)cascades of isoflavone accumulation induced by ABA-and NO-triggered cGMP in soybean sprouts under UV-B radiation was investigated.After UV-B treatment,gene expression of MAPK in 4-day-old soybean sprouts was 1.8 times higher than control.LY83583 and KT5823 inhibited UV-B radiation-induced gene expression of MAPK by 21%and 20%in 4-day-old soybean sprouts respectively.ABA,SNP and 8-cGMP could reverse the inhibition.ABA,SNP and 8-cGMP alone could also up regulate gene expression of MAPK.Therefore,ABA-and NO-triggered cGMP/PKG activated gene expression of MAPK under UV-B radiation.What’s more,U026(MAPKK inhibitor)inhibited UV-B radiation-induced isoflavone content and gene expression of CHS and IFS by 25%,19%and 31%in 4-day-old soybean sprouts,respectively.ABA,SNP and 8-cGMP could reverse the inhibition.Thus,MAPK,as the second biomessengers of cGMP/PKG,mediated isoflavone production under UV-B radiation.8.The mediation by Ca2+ of isoflavone accumulation in soybean sprouts under UV-B radiation was investigated.Ethylenebis(oxyethylenenitrilo)tetraacetic acid(EGTA)inhibited UV-B radiation-induced isoflavone content by 59%,activity of CHS and IFS by 47%and 24%,gene expression of CHS and IFS by 44%and 43%,protein expression of CHS and IFS by 18%and 27%in 4-day-old soybean sprouts,respectively.ABA,SNP and 8-cGMP could reverse the inhibition.Thus,Ca2+ mediated isoflavone production under UV-B radiation. |
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