Regulation Activity Of Plant Stem Cell And Telomere Length By Ginsenosides

An important goal for medicinal resource research is to produce Chinese materia medica best in quality. The main active conponents are mainly from secondary metabolites of meidicinal plant. Thus, many strategys are always employmented to increase the accumulation accounts of secondary metabolites for obtaining good quality products. However, many researches mainly focus on the usages of secondary metabolites, the physiological functions of those components in plant are still missing. So it is very intresting to see the function of the chemical constitunents in meidicinal plant. As Panax ginseng C. A. Mey is an herbaceous perennial plant and has an incredible lifespan of hundreds of years. Besides, ginseng has been widely used in several system diseases treatment, and the pharmacological activities of ginseng are mainly attributed to the presence of ginsenosides. The aim of present research is to study the physiological functions of ginsenosides.Ginseng is the dried root and rhizome of Panax ginseng C. A. Mey, which belongs to Araliaceae. It is one of the most wildly used Chinese herbal drugs in clinic. Ginseng has a usage history of 4000 years and is recorded firstly in Shennongbencaojing, and has tonic function to many systems of human body. The main active ingredients are ginsenosides, which belong to the terpenoids of dammarane- or oleanane-type. Ginsenosides have the functions of protecting central nervous and cardiovascular systems, regulating endocrine system, anticancer and enhancing immunity. As the contents of total ginsenosides increase continuously with the duration of cultivation, it is widely accepted that the longer time ginseng grows, the better quality it has. A wild ginseng can be valued for million yuan and has been a kind of scarce collection. Recent studies have shown that ginsenosides can regulate biological processes of stem cell, such as promoteing proliferation, differentiation, and repairing damaged cells, which provide scientific basis for ginseng’s anti-aging.Secondary metabolites are always taken as the main active components of crude drugs, and traditionally play important roles in adapting new environment, overcome- ing stress, and chemical defense of plant. However, more and more studies have revealed that those chemicals are also taken as indispensable part of plant growing regulation system, and play an important role in plant growth. Many factors accompany with the whole lifecycle of plant, and affect related biological processes, such as external environmental factors, biotic factor, and internal genetic character, related regulatory factors, plant hormones, active oxygen, stem cells, telomere and so on. The specialized regions in root and shoot tip called meristems, of which the center contains stem cells capable of self-renewal and differentiation into specific cell types, repair damaged cells. The root Stem Cell Nich （SCN） can control the initiate organ formation, reproductive growth and senescence of plant due to signals from surrounding cells. Telomere is DNA-protein structure that form protective cap at the end of eukaryotic chromosomes. Telomeres mainly involve in maintaining genomic stability and telomere length and they would shorten with each round of DNA replication in somatic tissues of most species due to the end replication problem. Once telomeres shorten to a critical length, a cell cycle checkpoint is triggered and cells enter a terminally none dividing state. Thus, telomere length is important for the stability of chromosome and cell division. Telomere is also taken as the molecular clock of DNA and a biomarker of aging in humans. As secondary metabolites of ginseng, ginsenosides can regulate stem cell fate and telomere length of human and animals. The regulation action of stem cell fate is similar in animal and plant. Thus, the present research will explore the regulation on stem cell and telomere by ginsenosides, and then reveal the effect of ginsenosides on plant growth regulation. The study can provide scientific basis for ginseng longevity and open up new a view for physiological functions of secondary metabolites.Based on the research background, the following experiments are implemented:1 Variation of ginsenosides along with planting yearsQualitative and quantitative analysis of ginsenosides are conducted using a UPLC-UV-MS method. Eight main ginsenosides in three tissues （leaf, rhizome and main root） and four parts （periderm, phloem, cambium and xylem） of ginseng aged 1-13 years are determined using a UPLC-PDA method. It is found that the contents of ginsenosides increased with cultivation years, causing a sequential content change of ginsenosides in an organ-specific manner. The accumulation amounts of total ginsenosides are the most in the leaf, and the amounts have fluctuant changes with the duration of cultivation. The contents of ginsenosides in the leaf samples vary from 36.64 to 59.03 mg/g, which indicate an approximate doubling in the ginseng samples grown from 1 to 13 years. Compared with the amounts of ginsenosides accumulate in leaf samples, the amounts in the rhizome samples show a downward trend after a first rise, followed by values varying from 10.76 to 65.30 mg/g within the studied 13 year growth cycles. In addition, it is worth noting that the rhizome has stable accumulation with the ranges of 23.09-31.88 mg/g from 2 to 7 years. The amounts of ginsenosides accumulated in rhizome samples increased sharply from 24.48 to 43.92 mg/g during the 7th and 8th growth year. Among the three detected tissues, the main root has the lowest contents of ginsenosides in 1-13 year-old ginseng, with a range from 16.27 to 31.14 mg/g. The amounts of eight active ingredients accumulate in the main root also follow a similar growth trend as those in leaf and rhizome extracts. Ginsenoside Rg1, Re and Rb1 are the major constituents in main root and rhizome. By contrast, the content of ginsenoside Rb1 is relatively low in leaf extracts, while ginsenoside Rgi and Re represent the dominant components. The contents of ginsenoside Ro are similar in the three tissues, and increase with the duration of cultivation. Besides, the amounts of ginsenosides accumulated in the periderm are 45.48 mg/g, which are more than twice as high compared with the other three parts.2 Screening active components that affecting the division of root stem cell from ginsenosides.At the tip of roots, the center of the root tip is the quiescent center （QC）, which is mitotically relatively inactive, and the surrounding cells are called stem cell with high mitotic activity. Thus, the stem cell nich （SCN） contains a small group cell of QC and stem cells that exhibit different proliferative activity. As the research on the SCN of ginseng is scarcely focused, and most researches of plant stem cell are studied in Arabidopsis thaliana. The active component affected plant stem cell is needed to be screened in Arabidopsis thaliana. Based on the chemical structure and contents, six ginsenosides （ginsenoside-Rgi, Rbi, Re, Ro, PPD and PPT） are chosen in the experiment. Three concentrations （50,100,150 μM） of ginsenosides are added into culture medium, and the seed germination is normal. However, the primary root length of Arabidopsis thaliana exposed to ginsenosides is inhibited, and the inhibition effect depends on the concentration. Only one ginsenoside of screened ginsenosides, GS1 treatment has the strongest inhibition effect of root length. Besides, the seedings exposed to ginsenosides show a significantly reduced number of root meristematic cells, which is defined by counting the number of cells from the cortical initial cell to the first elongated cell in the cortex layer. Consistently, the expression domain of CycB1;1:GUS, a widely used marker for the G2/M phase of the cell cycle, is reduced in the treated seedings, in which GS1 still has strongest effect. We next examine the cellular organization of the QC and its surrounding stem cells by GS1 treatment. It is found that the scope of the QC marker gene （WOX5:GFP; QC184:GUS） increased, suggesting GS1 promotes QC division. To assess the possible effect of ginsenoside GS1-induced QC division on stem cell maintenance, we examine the cell layer immediately below the QC, at the position of the CSCs. The J2341 marker, which is specifically expressed in the single layer of CSCs, expands to more than one cell layer upon GS1 treatment. However, the division behavior does not depend on the regulation of PLT or SCR/SHR pathway, in which the expressions of related genes are slightly changed by GS1 treatment.The results indicate that GS1 induce inhibition of primary root growth by decreasing meristematic activity. It also regulates the activities of stem cell niche and leads to division of QC and columella stem cell. However, the mechanism is still unknown. Genetic screening and map based cloning are explored to find the key gene.3 Genetic screening and map based cloning of key geneThe mutant insensitive to GS1 is screened from mutant population with about 980 mutagenesis. The 14th mutant is found resistant to GS1, named gsi （Ginsenoside insensitive mutant）. The homozygous gsi is then crossed to Ler ecotype to create a mapping population. Once the F2 generation of a mapping cross is available, the mutation is assigned a rough position on the genetic map by identifying linked 22 genetic markers. Next, two markers are found that are about 5% recombination apart and define a genetic interval containing the mutation. Utilizing these flanking markers, a large mapping population is screened for recombinants in the vicinity of the mutation. Finally, the genetic interval containing the mutation is narrowed down as much as possible by creating and analyzing new markers in the region of 60 kb, in which 10 genes are cloned and sequenced. The gene accession code of AT5G20950 is found having a mutational base in its CDS region, in which G is changed to A. Preliminary confirmation has been done by comparing the SALK line （SALK₀58396C） of the mutant gene, in which the phenotype is consistent with the screened mutant. Thus, the GSI gene （AT5G20950） plays an important role in participating inhibition of root length by GS1 treatment.4 The function confirmation of GH geneThe GH gene codes a glycosyl hydrolase, which includes 625 amino acids. Glycosyl hydrolase has two functional domains:Glyco_hydro₃ and Glyco_hydro₃C. The Glyco_hydro₃C domain is involved in catalysis and may be involved in binding beta-glucan, which is very important for catalytic reaction. Besides, the mutational position is just in the domain and may results in the deficiency of gene function. It is very interesting to see whether GS1 is hydrolyzed by glycosyl hydrolase. In order to confirm the possible hydrolysis sugar based from GS1 by glycosyl hydrolase, enzymatic reaction is carried out, in which glycosyl hydrolase is obtained by prokaryotic expression. The reaction is in NaAC buffer （pH 5.0） for overnight. The reaction products are analyzed by UPLC-MS, and an added ion peak is found in the chromatograph compared to the control group, suggesting a new component is produced. The new component is believed as a product of GS1 with loss a sugar by m/z value. Besides, the root length of gsi is inhibited by GS1-1 treatment. In conclusion, GS1 is first hydrolyzed to a new component GS1-1 by glycosyl hydrolase, and then inhibit the root length.5 Ginsenosides regulate the telomere lengths of ginsengThe main roots and leaves from 1-13 aged ginseng in four stages of seasonal growth, including leafing period, flowering period, red fruit period and withering period, were sampled. The overall average lengths of telomeric repeats in the studied samples were measured by the established PCR method. The consecutive 6 age classes of the samples were compared, and the results clearly shown that the variety of relative telomere lengths （RTLs） in main root decreased at first four years and then increased with age. The ginseng seeding taken as 1 year-old sample has longer telomere length and then the telomere lengths shorten in the three duration of cultivation. Significant differences are found in the 3- and 4- year-old ginseng, which have the shortest telomere length in the studied samples. The gradually elongated telomere lengths are observed in 5- and 6- aged ginseng samples, which also significantly increase compared with the shortest RTLs. The 1-6 aged ginseng samples of main root in four seasonal development stages show similar phenomenon. In order to clarify the varieties of RTLs with age, older aged ginseng root of 10-14 years and 15-30 years ginseng were obtained. Their RTLs were further measured and showed slowly extended along with years. Ginsenosides were also found affect RTLs of ginseng cell culture. The total ginsenosides could extend RTLs of cell culture in three concentrations, and the significantly longest telomere repeats was observed in the 5 μM ginsenosides medium.For the individual ginsenoside treatment, three components all significantly elongated the RTLs in low and middle concentrations （5,50 μM） treatment, while shorter telomere repeats were observed in 200μM ginsenoside. The most significant elongation of RTLs appeared in the middle concentration of ginsenoside Rbi treatment, while the effects were observed in 5 μM treatments for ginsenoside Rg1 and Re. Furthermore, the expression of telomerase gene was significantly up-regulated in the treatment samples. More specifically, the expression of telomerase gene was activated in 5 μM ginsenoside Rb1, and then had a decline in the higher concentrations, which was agreement with those of telomere lengths in the Rb1 treatment. The expression of telomerase gene was detected in those treated samples at the same time. Compared to the control group, the telomerase gene was up-regulated almost in all the samples exposed to the ginsenosides. In conclusion, the results indicated that the telomere lengths positively vary with age gives a new view that telomere length plays essential roles in regulating life-span of Panax ginseng, in which ginsenosides also involve in extending the TLs by activating of telomerase.Chemical defense is always taken as the main physiological function of secondary metabolites. However, the results from present study have shown that ginsenosides, as the secondary metabolites of ginseng, can affect plant growth by regulating plant stem cell nich and telomere length. As an herbal plant, ginseng can last a life-span of decades, in which the accumulation amounts of ginsenosides can increase along with the cultivation years. Telomere, as a biomarker of ages, elongates with the planting years in Panax ginseng, which is similar with the woody plants of longevity. Besides, ginsenosides contribute to the effect of telomere elongation. The telomere length is longest in plant root stem cell nich, and ginsenoside can affect the behivors of stem cell nich. In conclusion, ginsenosides, plant stem cell and telomere can together regulate the biological process of plant and may contribute to the longevity of Panax ginseng. The present research provides a new view for studying on the physiological function of secondary metabolites, in which plant stem cell and telomere are breakthrough points. The exploratory researches clarify the mechanism of secondary metabolites by molecular genetics strategy and provide a reference for the longevity of plant.