Investigation On The Mechanisms Of Efficient Biosynthesis Of Astaxanthin In Duced By Melatonin In Haematococcus Pluvialis

Astaxanthin has multiple unsaturated double bonds.This special molecular structure gives it superior anti-oxidation,neutralizing free radicals,and ability to scavenge reactive oxygen species?ROS?.Therefore,it has been widely used in health care products,cosmetics,food,and feed industries.Using various chemicals to trigger or enhance biosynthesis of astaxanthin have attracted significant attention since Haematococcus pluvialis is considered as the best natural source of astaxanthin.The mechanisms of action of melatonin?MLT?is not clearly understood although it exhibits pleiotropic and physiological function in numerous plant species.In this study,in order to study the effects of NO,ROS and SA on the gene expressions of the astaxanthin biosynthesis,the cultivation system of microalgae H.pluvialis LUGU will be reconstructed for highly efficient synthesis of astaxanthin using MLT as the chemical elicitor.Moreover,to elucidate the regulation mechanism of the astaxanthin biosynthesis induced by MLT,signal transduction mechanism of the astaxanthin biosynthesis will be clarified.Furthermore,this study will reveal the transformation mechanism between two antioxidative systems and astaxanthin associated with combined MLT application.The research results will be helpful to provide scientific basis and target for improving astaxanthin accumulation by the genetic engineering approach,and they will serve both theoretical and practical significance for increasing astaxanthin biosynthesis in the future research.The results are summarized as follows:?1?The astaxanthin content of the microalgae increased 2.36-fold after treatment with 10?M of melatonin.The maximal astaxanthin content achieved was 31.32 mg g^-1.The increased NO and SA production caused by melatonin occurred in parallel with the up-regulation of the expression of astaxanthin biosynthesis genes and the enhancement in astaxanthin biosynthesis in H.pluvialis LUGU.The analysis of fatty acid composition showed that melatonin stimulated the production of C16:0?palmitic acid?;thus,the percentage of astaxanthin esters was higher than that observed in the control algae.Furthermore,the inhibitors,namely,carboxy-PTIO,paclobutrazol,and N-acetyl-L-cysteine,significantly suppressed the levels of secondary messengers.Meanwhile,the role of melatonin in mediating the rise in astaxanthin content was prevented when the SA-and NO-dependent pathways were inhibited.These findings indicate that melatonin plays a physiological role in direct and indirect responses to abiotic stresses in H.pluvialis.?2?Exogenous melatonin improved stress resistance in H.pluvialis under nitrogen starvation and high light conditions.We conclude that exogenous melatonin treatment prevented the reactive oxygen species?ROS?burst and limited cell damage induced by abiotic stress;this effect involved the activation of several antioxidant enzymes and antioxidants.Astaxanthin,a major antioxidant in H.pluvialis cells,exhibited a 2.25-fold increase in content after treatment with melatonin.The maximal astaxanthin content was 32.4 mg g^-1.The functional roles of the NO-mediated mitogen activated protein kinase?MAPK?signalling pathway and cyclic adenosine monophosphate?cAMP?signalling pathway induced by melatonin were also evaluated.The results clearly indicate that cAMP signalling pathways are positively associated with microalgal astaxanthin biosynthesis.Additionally,the NO-dependent MAPK signalling cascade is activated in response to astaxanthin accumulation induced by melatonin,confirming that MAPK is a target of NO action in physiological processes.This work is the first to use H.pluvialis as in vivo model and documents the influence of melatonin on the physiological response to abiotic stress in this microalgae.Reveal the regulatory mechanism of"melatonin-signaling molecule?ROS,NO?-MAPK?cAMP?-astaxanthin synthesis".?3?Under stress conditions,melatonin induction two sets of defense systems in the cells of H.pluvialis,ie increased antioxidant enzymes?SOD,POD,CAT?activity and promoted the accumulation of astaxanthin through increased via signal transduction.The activity of POD,CAT and SOD in melatonin treatment group increased by 3.19-,1.45-and 1.35-fold,respectively.It is shown that under stress conditions,H.pluvialis increases the interrelationship and dynamic changes of two antioxidative systems to protect cells from damage,while the addition of exogenous melatonin can enhance the defense system of algal cells.Further improve the resistance and survival rate of H.pluvialis.In other words,MLT not only increase the accumulation of astaxanthin in H.pluvialis also inhibits the decrease of biomass under stress conditions.