Mechanism Of Hydrogen-rich Water Mediates Anthocyanin Accumulation In The Hypocotyls Of Radish Sprouts Under UV-A Radiation

Radish sprouts are important green sprouts.The application of an safe and efficient technology of environmental control in improving the nutritional quality of sprouts is very important for mainting a safe food supply.Hydrogen gas(H2)is an important gas signal molecule.In recent years,the application of hydrogen-rich water(HRW)to explore the physiological function of H2 in plants has become a new research hotspot.Ultraviolet light(UV)is an effective promoting factor in the biosynthesis of anthocyanin.However,the mechanisms of HRW on anthocyanin synthesis under UV-A remains elusive.In this study,'Yanghua' radish sprouts were used to explore the regulatory role of H2 in the accumulation of anthocyanin under UV-A.the main results are shown as follows:1.Hydrogen-rich water(HRW)was used to study the effect of H2 on the accumulation of anthocyanin in radish buds under different spectral wavelengths.The results showed that the four anthocyanins were identified in radish sprouts,which were cyanidin,cyanidin-3-glucoside,cyanidin-3-rutinoside-5-glucoside and petunidin-3,5-diglucoside.Compared with white light,blue light significantly increased the content of petunidin-3,5-diglucoside,and blue light+HRW further increased the content.Compared with other light treatments,UV-A significantly increased the content of cyanidin-3-rutinoside-5-glucoside and cyanidin-3-glucoside,and those two anthocyanin content was further increased when HRW was co-treated with UV-A.Besides,The total anthocyanin content was the highest in UV-A and HRW treatment.HRW increased the activity of PAL,CHS and UFGT under UV-A,and also promoted the expression level of key genes,such as RsPAL,RsCHS,RsUFGT,RsF3'H,RsDFR and RsANS.The antioxidant capacity of anthocyanin extract of radish sprouts increased with the increase of total anthocyanin content.In general,HRW promoted the gene expression and enzyme activity of anthocyanin biosynthetic-related key enzymes under UV-A,and enhanced the antioxidant capacity of radish sprouts anthocyanin extracts.UV-A and HRW cotreatment was the most favorable treatment to improve anthocyanin content.2.Methyl Jasmonate(MeJA)is an important plant hormone that participates in the regulation of anthocyanin synthesis.However,the role of MeJA in the regulation of anthocyanin synthesis under HRW treatment remains unclear.Compared with the control,UV-A significantly increased H2 release and endogenous H2 content in hypocotyls of radish sprouts.Compared with UV-A,UV-A+HRW treatment farther increased the H2 release and endogenous H2 content in the hypocotyls of radish,and MeJA did not affect the production of H2.Under UV-A radiation,HRW significantly increased the level of endogenous MeJA and the key enzyme(LOX)activity of MeJA biosynthesis,and promoted the expression of RsLOX,RsAOS and RsAOC in MeJA synthesis pathway.The promotion effect of HRW on the synthesis of anthocyanin was inhibited by the addition of MeJA synthesis inhibitor SHAM,and the expression level of RsCHS?RsCHI?RsDFR and RsUFGT was inhibited.Similar to MeJA treatment,HRW promotes ubiquitination degradation of RsJAZ1 protein,a negative regulator of jasmonic acid,under UV-A radiation.The results showed that HRW could increase the content of endogenous HZ and enhance the endogenous MeJA level under UV-A,and the endogenous MeJA promoted the ubiquitination of the RsJAZ1 protein,which inhibited the inhibition of anthocyanin synthesis by RsJAZ1 and ultimately promoted the synthesis of anthocyanin.3.The results of our previous studies show that HRW promotes the synthesis of hypocotyl anthocyanins from radish sprouts induced by UV-A,but the molecular mechanism is not clear.The promoting effect of HRW on the accumulation of anthocyanin in radish sprouts mimicking the inducing effects of CaC12.Exogenous addition of calcium chelator EGTA and IP3 synthesis inhibitor Neomycin could partially reversed the effects of HRW on the biosynthesis of anthocyanin.The above results indicate that IP3-sensitive calcium channels may be involved in the regulation of anthocyanin synthesis by HRW under UV-A radiation.Further experiment showed that the enzyme activity of PAL and CHI was promoted by HRW,and this phenomina disappeared under Neomycin treatment.Similarly,the expression of key genes in anthocyanin synthesis was promoted by HRW,while Neomycin treatment inhibited the expression of those key genes.Meanwhile,CaM antagonists W7(N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide)and TFP(trifluoperazine)inhibited the HRW-promoted biosynthesis of anthocyanin under UV-A.While the CaM antagonist W5(N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride)had no effect on the content of anthocyanin.In general,HRW may regulate the synthesis of anthocyanins through the IP3-dependent calcium ion signaling pathway under UV-A.4.Glutathione S-transferase(GSTs)plays an important role in the accumulation of anthocyanins and proanthocyanidins in plants.GSTs is a multifunctional protease that plays an important role in the accumulation of anthocyanins and proanthocyanidins.In this study,we used RT-PCR to clone the radish RsGSTF12 gene.The results showed that the expression profiles of RsGSTF12 gene in radish sprouts were consistent with the accumulation profiles of anthocyanin in different tissues and external stimuli.The expression of RsGSTF12 could be induced to a different degree by UV-A,UV-A+HRW,melatonin,hematin,sucrose and MeJA.In addition,compared to UV-A,UV-A+HRW significantly up-regulated the expression of RsGSTF12.The analysis of predicted amino acid sequences and phylogenetic trees showed that RsGSTF12 was closely related to AtGSTF12(TT19).Radish RsGSTF12 encodes a 26.13 KD protein that may have anthocyanin transport function.Subcellular localization analysis showed that RsGSTF12 was localized in the nucleus and cytoplasm.In vitro analysis showed that the purified recombinant RsGSTF 12 protein increased the water solubility of cyanidin(Cya).The above results provide a basis for futher study of the function of RsGSTF12 in anthocyanin accumulation and transport.Meanwhile,the mechanism of HRW promoting the accumulation of anthocyanin in UV-A was also further explained.