Study On The Screening Of Melatonin-producing Endophytic Bacteria From Grapevine Roots And Its Synthetic Pathway

As a kind of tryptophan derivative widely found in animals,plants and single cell organisms,melatonin has attracted much attention in recent years because of its unique antioxidant and physiological regulation functions.The use of exogenous melatonin not only promote plant seed germination,root development,flowering and fruit ripening,but also prevent leaf senescence and improve plant tolerance under biotic and abiotic stress.Previous studies have shown that abiotic stress can improve the synthesis of endogenous melatonin in plants,which is due to the fact that plant-generated reactive oxygen species（ROS）activate biosynthesis of plant melatonin.Under natural conditions,the roots of terrestrial plants gather rich endophytic microorganisms that interact with host plants and regulate plant growth and development.Endophytes are the beneficial components of plant growth and development,and they often have the ability to synthesize plant hormones and are able to alter the host’s endogenous hormone levels;however,there is no evidence that endophytes are capable of synthesizing melatonin,and affects the physiological metabolism of the host plant.In this study,the endophytic bacteria in the roots of four grape varieties were isolated and identified,and the strains with melatonin synthesis ability were screened.Stable isotope labeling combined with UPLC-MS/MS were used to study bacterial melatonin biosynthetic pathway.Prokaryotic expression and gene knockout were used to verify the function of L-phenylalanine hydroxylase involved in bacterial melatonin synthesis.The main findings are as follows:1.Separation of endophytic bacteria from the roots of “Cabernet Sauvignon”,“Red Globe”,“Summer black” and China Wild Vitis amurensis “Changbai 9” were carried out by traditional isolation and identified with 16 S rDNA sequencing.The ability of melatonin production was determined by UPLC-MS/MS.The results showed that there were 6 genera and 15 species of endophytic bacteria isolated from the roots of four grape varieties.Five strains of bacteria,Bacillus thyloliquefaciens SB-9,Bacillus thuringiensis CS-9,Pseudomonas fluorescens RG11,Agrobacterium tumefaciens CS-30 and Variovorax soli VA10,had the ability of melatonin synthesis at a concentration between 0.15–7.75 ng/10122.The endophytic bacteria,Bacillus amyloliquefaciens SB-9 and Pseudomonas fluorescens RG11,which had the strongest synthesis ability of melatonin,were used to infect the root system of different grape varieties,and to explore the effect of melatonin endophytic bacteria on promoting plant endogenous melatonin levels and the potential application of enhanced host plant stress resistance.The results showed that B.amyloliquefaciens SB-9 and P.fluorescens RG11 significantly increased the fresh weight,chlorophyll content,root length and lateral root number of the seedlings,and showed excellent growth promoting effect.In addition,the colonizationof B.amyloliquefaciens SB-9 and P.fluorescens RG11 significantly increased the contents of endogenous melatonin and its precursors,but reduced the transcriptional evels of the tryptophan decarboxylase gene（Vv TDCs）and serotonin（VvSNAT）,and relieved the adverse effects of salt and drought stress by reducing the production of malondialdehyde（MDA）and reactive oxygen species in the roots.3.The stable isotope tracer method combined with UPLC-MS/MS were used to study the composition of the intermediate metabolites in P.fluorescens RG11 melatonin-synthesis.The results showed that,except for ¹⁵N-tryptamine,¹⁵N-5-hydroxytryptophan,¹⁵N-5-hydroxytryptamine,¹⁵N-N-acetyl-5-hydroxytryptamine and¹⁵N-melatonin were detected in the bacterial solution,indicating that the synthetic pathway of bacterial melatonin is similar to animal’s.The first step in the synthesis of melatonin is that the ¹⁵N-L-tryptophan carbon skeleton is hydroxylated to form 5-hydroxytryptophan,suggesting that phenylalanine hydroxylase may be responsible for the strain’s tryptophan hydroxylation.In addition,melatonin synthesis capacity of P.fluorescens RG11 was highest in the early growth period,but sharply declined in the late culture,suggesting that melatonin in bacteria may be used as a growth factor or as a protective factor for the removal of ROS in medium.4.The relationship between the prokaryotic phenylalanine hydroxylase（PAH）and the amino acid hydroxylase family was analyzed,and the PAH enconding gene of P.fluorescens RG11 was cloned and the PAH protein was obtained by prokaryotic expression.The results showed that PAH protein sequence of P.fluorescens RG11 had a high amino acid sequence similarity to that of animal amino acid hydroxylase.In vitro activity test showed that the purified recombinant PAH protein has both tryptophan and phenylalanine hydroxylase activity,hydroxylating of L-tryptophan into 5-hydroxytryptophan,and Km and V_max values were 2575μM and 0.834μmol 5-Hydroxytryptophan/min/mg protein,but showed lower affinity and enzymatic activity than L-phenylalanine hydroxylation.5.The phhA gene of P.fluorescens RG11 was knockout to generate a ΔPhhA mutant strain by homologous recombination.The N15 isotope tracer method and UPLC-MS/MS were used to further study the relationship between bacterial phenylalanine hydroxylase andmelatonin-producing.The results showed that phhA gene knockdown significantly reduced the yield of 5-hydroxytryptophan and subsequent metabolites.The ΔPhhA strain did not detect melatonin throughout the culture period,indicating that PAH is responsible for the synthesis of 5-hydroxytryptophan in RG11 strain and plays an important role in the synthesis pathway of melatonin.