Analysis Of The Heavy Metal Content Of Polygonatum Cyrtonema Hua And The Effect Of Melatonin On It Mitigation Of Lead Stress In Tissue-culture Seedlings

Polygonatum cyrtonema Hua is a perennial herb in the genus Polygonatum of the family Liliaceae.It is a dual-use plant,rich in polysaccharides,saponins,flavonoids and other active ingredients,and has the effect of improving human immunity,nourishing Yin and moistening the lungs.Heavy metals（Pb,Cd,etc.）were found to be exceeded in soils of Fujian Province,while Polygonatum cyrtonema Hua extract was found to be exceeded in different areas.heavy metal analysis of Polygonatum cyrtonema Hua extract in Fujian Province has not been reported yet.Therefore,this study was conducted to analyze the heavy metal content of Polygonatum cyrtonema Hua in different areas of Fujian Province to provide data support for the safe consumption of Polygonatum cyrtonema Hua in Fujian Province.The growth and physiological parameters of Polygonatum cyrtonema Hua seedlings treated with different concentrations of Pb were measured to investigate the effects of Pb treatment on Polygonatum cyrtonema Hua seedlings.the optimal concentration of MT（Melatonin,MT）treatment on the growth and physiological parameters of Polygonatum cyrtonema Hua seedlings under Pb stress was also investigated.The transcriptome and metabolome differences between Pb stress and MT treatment were also analyzed to reveal the mechanism of MT on Pb stress mitigation in Polygonatum cyrtonema Hua,and the gene families of heavy metal-associated plant proteins（HIPPs/HPPs）in Polygonatum cyrtonema Hua were identified and analyzed for expression..The aim was to provide a theoretical basis for an in-depth investigation of the role of melatonin in alleviating Pb stress and its regulatory mechanism in Polygonatum cyrtonema Hua.The main results are as follows:（1）Analysis of heavy metal content of Polygonatum cyrtonema Hua in different regions of Fujian ProvinceIn this study,the analysis of heavy metals（cadmium,copper,arsenic,mercury and lead）in different areas of Fujian Province found that the levels of all five heavy metals were within thenormal limit concentrations.To determine the safety of the drug,a safety risk assessment was conducted on Polygonatum cyrtonema Hua,and risks with a hazard index>1/exposure limit≤1 should be of concern,it was found that the heavy metal content of Polygonatum cyrtonema Hua in Fujian Province was within the safe range,but the risk of Pb was high in 16 locations.except for Shaowu Yanshan wild and artificial,the risk of mercury was high in 14 locations.arsenic was high in 6 locations,namely Ninghua Anyuanwild,Dehua Chishui wild,Changting Tufang artificial,Changting Zhuotian artificial,Changting Meijing artificial and Zherong Yingshan artificial.（2）Effects of different concentrations of Pb treatment on tissue-culture seedlings of Polygonatum cyrtonema HuaIn analyzing heavy metal content and risk assessment,the highest health risk of Pb in Polygonatum cyrtonema Hua was found in Fujian Province.Therefore,Polygonatum cyrtonema Hua was treated with different concentrations of Pb chloride in a histoculture system to investigate the effect on the production of Polygonatum cyrtonema Hua tissue-culture seedlings.Firstly,the seedlings of Polygonatum cyrtonema Hua were analyzed for growth index.It was found that after 1 mM of PbCl₂treatment,their leaves shrank,yellowed severely,and their biomass decreased.Further analysis of chlorophyll content,SOD and POD in Polygonatum cyrtonema Hua under Pb treatment revealed that chlorophyll content,SOD and POD were significantly down-regulated in Polygonatum cyrtonema Hua under 1 mM PbCl₂treatment.In addition,analysis of polysaccharides and flavonoids in the histoculture seedlings of Polygonatum cyrtonema Hua revealed that Pb treatment promoted polysaccharide accumulation in Polygonatum cyrtonema Hua.Meanwhile,it had less effect on flavonoid content.Therefore,1 mM PbCl₂significantly inhibited the growth,chlorophyll content,SOD and POD activities of Polygonatum cyrtonema Hua,which caused the stress to Polygonatum cyrtonema Hua tissue-culture seedlings.（3）Mitigating effects of different concentrations of MT treatment on Pb stress in Polygonatum cyrtonema HuaThe treatment of 1 mM PbCl₂was found to cause significant stress to Polygonatum cyrtonema Hua tissue-culture seedlings.Therefore,on the basic of 1 mM PbCl_2,different concentrations of MT（50,100,150 and 200μM）were further added to the subculture medium of Polygonatum cyrtonema Hua extract to investigate the optimal concentration of MT in alleviating Pb stress.The results showed that the treatment with 200μM melatonin effectively alleviated the wilting,leaf yellowing,biomass and other growth states of the Polygonatum cyrtonema Hua seedlings.It was found that 200μM of melatonin could significantly up-regulate the chlorophyll content of the treated seedlings,SOD and POD activities were also significantly increased.At the same time,the polysaccharide and flavonoid contents were significantly increased under 200μM MT treatment.Thus,200μM MT had the most significant alleviating effect on the growth,chlorophyll content,SOD and POD activities,polysaccharides and flavonoids content of Polygonatum cyrtonema Hua under Pb treatment.（4）Combined analysis of exogenous MT regulation of Pb stress mitigation in Polygonatum cyrtonema HuaThe nuclear damage was observed in different days of Polygonatum cyrtonema Hua tissue-culture seedlings,it was found that the combined treatment of 1 mM PbCl₂with 200μM MT（MT4）reduced the nuclear damage caused by Pb stress in days.Therefore,further transcriptome sequencing analysis was carried out on the tissue-culture seedlings of Polygonatum cyrtonema Hua（CK）,tissue-culture seedlings under 1 mM PbCl₂treatment（Pb4）and tissue-culture seedlings of MT4.A total of 61.43 Gb of high quality reads were obtained,and the comparative rates ranging from 80.92 to82.86%.4673,7368 and 4181 differentially expressed genes were screened in the treatment groups of CK-Pb4,CK-MT4 and Pb4-MT4for pairwise comparisons,respectively.KEGG enrichment analysis revealed that the differential expression genes were most significantly enriched in protein processing and zeatin biosynthesis by the endoplasmic reticulum,which might have important regulatory roles in the mitigation of Pb stress in Polygonatum cyrtonema Hua.The expression ofβ-regulatory subunits,mitogen-activated protein kinase,peroxidase in AMPK and MAPK signaling pathways were significantly up-regulated under MT4 treatment.The expression of Hydroflavonol4-reductase/flavonol4-reductaseand Hydroxycinnamoyl transferase in flavonoid biosynthesis pathway were significantly up-regulated under MT4 treatment.4 members of HIPPs/HPPs which belong to the heavy metal related gene family were significantly up-regulated under MT4 treatment.Further metabolomic analysis of CK,Pb4 and MT4-treated Polygonatum cyrtonema Hua resulted in the detection of 1649metabolites,including 12 groups of amino acids and their derivatives,phenolic acids,nucleotides and their derivatives,flavonoids,quinones,lignans and coumarins.217,267 and 131 differential metabolites were screened in pairwise comparisons between treatment groups of CK-Pb4,CK-MT4 and Pb4-MT4,respectively.KEGG enrichment analysis indicated that the differential metabolites were most significantly enriched in the flavonoid biosynthesis,tryptophan metabolism,and flavonoids were significantly up-regulated under MT4 treatment.Joint analysis of transcriptome and metabolome showed that the tryptophan metabolic and the flavonoid biosynthesis pathway were key pathways for MT to alleviate Pb stress in Polygonatum cyrtonema Hua.（5）Identification and expression regulation of HIPPs/HPPs gene family based on the transcriptome of Polygonatum cyrtonema Hua.Analysis of transcriptomic data from Polygonatum cyrtonema Hua suggested that 4 members of HIPPs/HPPs which belong to the heavy metal related gene family were significantly up-regulated under MT4 treatment.Therefore,identification and expression profiling of the HIPPs/HPPs in Polygonatum cyrtonema Hua was carried out.Based on different transcriptomic data of Polygonatum cyrtonema Hua,68 PcHIPPs and 43 PcHPPs members were identified,respectively.Subcellular localization analysis showed that 73PcHIPPs/PcHPPs were distributed in the nucleus.The results showed that the expression of PcHIPPs/PcHPPs was up-regulated in the rhizomes of Polygonatum cyrtonema Hua,and most of the PcHIPPs/PcHPPs showed significant down-regulation at 48 h of salt stress treatment.qRT-PCR showed that 1 mM PbCl₂treatment inhibited the expression of PcHPPs in the rhizomes of Polygonatum cyrtonema Hua.Most of the PcHIPPs/PcHPPs were inhibited by 1mM PbCl₂treatment,and the expression of the PcHIPPs/PcHPPs increased after the addition of 200μM MT to the same treatment.