Study On The Function Of Arabidopsis Melatonin In Response To UV-B Stress And The Expression Of Its Synthase

As an crucial indole signaling substance in animals and plants,melatonin plays an important role in plants.In response to UV-B stress,plants have evolved sophisticated mechanism to regulate its growth and resist damage.In order to clarify the role of melatonin under UV-B stress and the function of three melatonin synthase in Arabidopsis,the overexpression lines of melatonin synthase including ASMT,COMT and SANT,T-DNA mutants of SNAT and GUS transgentic lines including SANT,COMT and ASMT were used in this study.At first,the temporal and spatial expression characteristics of three melatonin synthetase in Arabidopsis thaliana were analyzed by GUS histochemical staining and qRT-PCR.Secondly,after UV-B radiation treatment,qRT-PCR was used to analysis the relative expression level of ASMT,COMT and SANT.And the accumulation of melatonin was determined by ELISA Kit.Thirdly,qRT-PCR technique was used to detect the relative expression of UV-B signaling pathway genes and UV-B effector genes;Besides,many physiological experimental method were used in this study,such as DAB staining,NBT staining,H₂O₂ quantification,MDA quantification and Electrolyte leakage assay.Our results suggested thatthat melatonin is an important signals in process of Arabidopsis respond and resist to UV-B radiation.The main research results are as follows:?1?The expression patterns Analysis of three Arabidopsis melatonin synthase genes by histochemical staining and qRT-PCR technique revealed that SNAT is highly expressed in flower organs and pods of Arabidopsis thaliana;COMT not only expresses high in vegetative growth stage of Arabidopsis,but also expresses high in reproductive growth stage;ASMT is mainly expressed in roots and floral organs.?2?To address the function of melatonin in UV-B stress,we constructed binary expression vector of pRI101-SNAT?pRI101-COMT and pRI101-ASMT,respectively.The recombinant plasmid was introduced into A.tumefaciens GV3101 by freeze-thaw method.A.tumefaciens mediated transformation of Arabidopsis was performed essentially according to the protocol 16,8,20 and 9 independent ASMT,COMT,SNAT1 and SNAT3 transgenic lines were obtained by kanamycin selection and PCR of genomic DNA,respectively.Subsequently,qRT-PCR showed,compared to wide-type Col-0,mRNA signals of transgenic lines are markedly intensified.Finally,the melatonin accumulation of transgenic lines were detected by ELISA,and the content of melatonin in transgenic lines are increased from 1.5 to 2 times.SNAT homozygous mutant?SALK₀32239?was gained by genotyping tests.Compared with wide-type Col-0 length of roots?4.6±0.16cm?,the roots length of snat is 3.47±0.25cm,SNAT1-OE is 5.2±0.175cm and SNAT3-OE is 5.3±0.136cm.These results certified SNAT affect the development of root.The different lines?SNAT1-OE?SNAT3-OE?Col-0 and snat?placed under 313nm UV-B.qRT-PCR results indicated,the mRNA levels of SNAT?COMT and ASMT were greater than Col-0 or snat at 2h.ELISA assay manifested,the accumulation of melatonin in SNAT1,SNAT3 overexpressed plants were 1 and 2 times higher than in Col-0,and 3 and 3.2 times higher than snat at 4h,respectively.Interestingly,Compared with SNAT1 transgenic lines,the abundance of SNAT,COMT and ASMT of SNAT3 transgenic lines are reinforce and the levels of melatonin of SNAT3 are strengthening.?3?We detected the expression of UV-B signaling pathway-related genes by qRT-PCR.The results show that,compared with Col-0,the relative expression of HY5in SNAT overexpressing plants increased significantly,while the relative expression levels of COP1,HYH,RUP1 and RUP2 were also increased;Meanwhile,the relative expression level of HY5 was the lowest in snat mutants.The experimental results show that the SNAT gene can affect the UV-B signaling pathway,and the relative expression of the downstream effector genes APX,SAPX,CAT1 and CSD1 is increased sequentially,which leads to the decrease of ROS content in the plant organism.The results of DAB staining,NBT staining and H₂O₂ content determination showed that the ROS content in the SNAT overexpressed transgenic plants was lower than Col-0 and snat at 4h and 6h.The MDA content showed that at4h and 6h,compared with Col-0 and snat,was lower in SNAT overexpressing transgenic plants.The results of Electrolyte leakage assay showed that the conductivity values of SNAT overexpressing transgenic plants were less than Col-0and snat at 2h,4h and 6h.These results indicate that an increase in melatonin content in Arabidopsis increases the plant's perception of UV-B radiation and increases resistance.SNAT3-OE and SNAT1-OE are alternatively spliced transcripts of SNAT.Meanwhile,the SNAT3-OE strain of SNAT was more resistant to UV-B than SNAT1-OE.