Mechanisms Involved In UV,Hydrogen Gas And Nitrogen-Regulated Anthocyanin Biosynthesis In Radish Sprouts

The biosynthesis and accumulation of anthocyanins in plant tissues could be significantly induced by UV-B irradiation.UVR8 is the receptor of UV-B,so it may be involved in this UV-B-induced anthocyanin biosynthesis.When plants are irradiated by UV-B,H2O2 will accumulate in plants,and NO was reported as the downstream signal of H2O2.Therefore,in the regulation of anthocyanin biosynthesis,there may be interaction between UVR8,H2O2 and NO.UV-A could also induced the accumulation of anthocyanins,and in this process ROS synthesis was enhanced.Recently,many researches have reported the role of H2 in alleviation of detrimental effects of aboitic stess(Cd,NaCl)on plant growth,development and seed germination.Thus,H2 may also be involved in UV-A-induced anthocyanin accumulation.While the researches about roles of H2 on plant physiology are still very limited,and it is still uncertain that whether there is a pathway for sensing and transporting H2 signal in plant tissues.The enhancement of anthocyanin biosynthesis and accumulation in plant tissues could also be induced by the macronutrient depletion in the growth environment,and nitrogen(N)was reported as the main factor in this process.Sugar exogenously addition,especially sucrose,could stimulate the anthocyanin biosynthesis.As the balance of N and carbon(C)metabolism influences most plant developmental processes,maybe there was a interaction between N and sugar in modulating anthocyanin biosynthesis.To explore the above questions,we performed relevant experiments using radish sprouts as the experimental material and got the following results:1.The cross talk among hydrogen peroxide(H2O2),nitric oxide(NO)and UV RESISTANCE LOCUS8(UVR8)in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated.The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of RsUVR8,and a similar trend appeared in radish sprouts subjected to cadmium,chilling and salt stresses regardless of light source.However,these responses disappeared under dark exposure.These results suggest that abiotic stress-induced anthocyanin accumulation and RsUVR8 expression were light-dependent.Moreover,abiotic stresses all enhanced the production of H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and RsUVR8 transcription,while these increases were severely inhibited by addition of dimethylthiourea(DMTU,a chemical trap for H2O2).It seems to suggest that H2O2 played an important role in the anthocyanin biosynthesis.Furthermore,addition of 0.5 mM sodium nitroprusside(SNP,a NO-releasing compound)substantially induced the anthocyanin accumulation,and H2O2-induced anthocyanin accumulation and RsUVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-l-oxyl(PTIO,a NO scavenger),which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes.All these results demonstrate that both H2O2 and NO are involved in UV-B-induced anthocyanin accumulation,and there is a crosstalk between them as well as a classical UVR8 pathway.2.The aims of the study were to investigate whether hydrogen gas(H2)was involved in regulation of anthocyanin biosynthesis in two contrasting radish(Raphanus sativus L.)varieties(low[LA]and high[HA]level of anthocyanin)under UV irradiation.The results showed that hydrogen-rich water(HRW)significantly blocked the UV-A-induced increase of H2O2 and O2·-accumulation,and enhanced the UV-A-induced increase of superoxide dismutase(SOD)and ascorbate peroxidase(APX)activities in LA and HA.Furthermore,UV-A-induced increase of anthocyanin and total phenols was further enhanced only in HA sprouts cotreated with HRW.LC-MS/MS analysis showed that five anthocyanidins existed in HA sprouts,but only two in LA sprouts.Meanwhile,the cyanidin was the most abundant anthocyanidin in HA,and the cyanidin was 2-fold higher cotreated with HRW than UV-A.Molecular analyses showed that the anthocyanin biosynthesis-related genes were upregulated significantly in both HA(in particular)and LA sprouts treated with HRW plus UV-A.These data imply that HRW reestablishes reactive oxygen species homeostasis in both LA and HA,but exerts different effects on anthocyanin accumulation between them under UV-A.3.Hydrogen is the most abundant element in the universe,and H2 has been reported to act as a novel beneficial gaseous molecule in plant adaptive responses.Radish sprouts are popular because they contain substantial amounts of antioxidants and health-promoting compounds,such as anthocyanin and glucosinolates.Although radish sprouts accumulated more anthocyanin under UV-A after treatment with hydrogen-rich water(HRW),the molecular mechanism responsible is still elusive.To explore these mechanisms,RNA-seq analysis was used.Four cDNA libraries from radish sprout hypocotyls were constructed,and a total of 14,564 differentially expressed genes(DEGs)were identified through pairwise comparisons.By GO and KEGG analysis,these unigenes were found to be implicated in light signal perception and transduction,starch and sucrose metabolism,photosynthesis,nitrogen metabolism and biosynthesis of secondary metabolites.The MYB-bHLH-WD40 complex accounted for the majority of the TFs found to be involved in anthocyanin biosynthesis,and levels of transcripts for this complex were in accordance with the anthocyanin concentrations observed.In addition,other TFs(such as NAC,bZIP and TCP)might participate in H2-promoted anthocyanin biosynthesis.Furthermore,the signaling processes of plant hormones,MAPKs and Ca2+ might be involved in H2-promoted anthocyanin biosynthesis under UV-A.The expression patterns of 16 selected genes were confirmed using qRT-PCR analysis.Taken together,the results of this study may expand our understanding of H2-promoted anthocyanin accumulation under UV-A in plants.4.Effects of nitrogen(N)deficiency and sucrose addition on regulation of anthocyanin biosynthesis and their relationship were investigated in this study.Radish sprouts subjected to N deficiency had 50%higher anthocyanin accumulation than when grown in Hoagland solution(a nutrient medium with all macronutrients).The contents of endogenous soluble sugars(sucrose,fructose and glucose)in the hypocotyls were also markedly increased by N limitation,with sucrose showing the highest increase.Inhibition of carbohydrate biosynthesis by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU)also eliminated N deficiency-induced anthocyanin accumulation.The latter was further supported by the expression of anthocyanin biosynthesis related genes and decreased activities of nitrate reductase in the presence of sucrose.Together our results indicate that N deficiency-induced anthocyanin accumulation was,at least partly,dependent on the increase of the soluble sugar,especially Suc.This work is the first comprehensive study on relationship between N deficiency and sugar content on anthocyanin accumulation in the hypocotyls of radish sprouts.