Introduction

Ginseng, which is the root of Panax ginseng C.A. Meyer (Araliaceae), has been used in Chinese medicine for thousands of years as a stimulant and dietary supplement1. In European and American countries, ginseng phytomedicines have been used to increase physical and mental performance, provide resistance to stress and disease and prevent exhaustion for decades2. Ginseng plants begin flowering in their fourth year and the roots can live for hundreds of years after maturing at 4–6 years of age. The older the root, the higher its medicinal value because of the higher concentration of ginsenosides, which are the active chemical compounds in ginseng3,4. However, chemical analyses often require gram quantities of dried ginseng material and it is difficult to extract such quantities while leaving the ginseng intact; thus, chemical analysis greatly decreases the ginseng's value. Therefore, effective methods for identifying the age of ginseng roots are urgently needed to improve quality control and protect the interests of ginseng consumers.

Telomeres, which are specialized structures at the physical ends of eukaryotic chromosomes that consist of highly conserved, repeated DNA sequences5,6, shorten with each round of DNA replication7,8,9,10 because DNA polymerases cannot completely replicate linear DNA molecules. In gymnosperms, telomere length can be used to predict the future replicative capacity of cells11,12. Highly significant correlations between telomere length and age have been observed in humans8,13, Australian sea lions14, martins and dunlins15 and different stages of barley16. Therefore, telomere shortening can be used as a marker of cell replication and aging. Telomerase activity has been detected in plants using a polymerase chain reaction (PCR)-based telomerase repeat amplification protocol (TRAP) assay17. Telomerase appears to be developmentally regulated in plants, which is similar to what occurs in humans18. These reports indicate biological correlations between telomere length and age. However, plant telomeres are maintained by telomerase. Telomere lengths remain stable in tomato leaves19, whereas they change cyclically, lengthening and shortening with age, in the needles of Pinus longaeva20. After the first plant telomere sequence was cloned from Arabidopsis21, nearly all plant telomeres were found to consist of the heptanucleotide repeat (TTTAGGG)n22,23. Arabidopsis-type repeats have also been found in P. ginseng24. However, researchers have not yet ascertained whether Arabidopsis-type repeats are located in telomeres or their relationship with age.

In this study, we combined traditional identification methods and measurements of telomere length in ginseng plants of known age. Preliminary investigations indicated that telomere length was slightly positively correlated with the age of the ginseng plant. Analysis of telomerase activity in different parts of the plant further revealed that the main root was the most active meristematic region. Therefore, we used this tissue to evaluate telomere length. Determination of telomere terminal restriction fragment (TRF) lengths in P. ginseng specimens of different ages demonstrated that the telomeres in the main roots showed a significant increase in TRF length with plant age that could be used for age estimation for 2–8 years.

Results

Fluorescence in situ hybridization to determine telomere sequences

The telomeres of most higher plant species are composed of the repeated sequence (TTTAGGG)n. To investigate P. ginseng telomeres comprising the same repeat, we using the complementary end digoxigenin-labeled, telomere-specific oligonucleotide (CCCTAAA)3 as a probe to perform in situ hybridization. Hybridization signals visualized as green fluorescence demonstrated that Arabidopsis-type telomeric sequence repeats, (TTTAGGG)n, were located in the chromosomes of P. ginseng (Fig. 1).

Figure 1
figure 1

In Situ Localization of TTTAGGG Telomeric Motifs on P. ginseng Chromosomes.

The the digoxigenin–dUTP nick tag sequence (CCCTAAA)5 telomeric probe was hybridized with adventitious root of P. ginseng metaphase chromosomes and counterstained with propidium iodide.

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Growth rings in the roots of P. ginseng from Ji'an

The paraffin sections of P. ginseng rhizomes of different ages collected from Ji'an revealed distinct growth rings in the xylem of secondary roots and the number of growth rings in the main root was consistent with an age of 1–6 years (Fig. 2). However, microscopy analysis showed that the growth rings of the ginseng specimens did not precisely reflect age after 6 years.

Figure 2
figure 2

The growth rings in the ginseng root of 1 ~ 6 years.

(A), (B), (C): 1 year ginseng root, 2 year ginseng root, 3 year ginseng root, Bar = 1000 μm; (D), (E), (F): 4 year ginseng root, 5 year ginseng root, 6 year ginseng root, Bar = 500 μm.

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Telomeric activity of different ginseng tissues

A representative TRAP analysis image that was used to quantify telomerase activity is shown in Fig. 3. Average telomerase activities in various tissues and different stages of plant development were assayed using TRAP and the results indicated that the main root showed the highest average telomerase activities of all of the examined tissues. Because telomerase can lengthen telomeres and the activity of telomerase may be correlated with age, the main roots were used for further analyses.

Figure 3
figure 3

Developmental Regulation of Telomerase Expression in 5 years P. ginseng.

Telomerase activities in various tissues and different stages of plant development were assayed by TRAP, using 47F as the forward primer and PTelC3 as reverse primers.Lane1: tap root; Lane2: leaves; Lane3: stems; Lane4: root tips; Lane5: seeds.

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Tangential cryo-sectioning of 400 μm sections of samples from 5-year-old P. ginseng tissues, followed by densitometric quantitation of telomerase activity (in relative units), revealed the highest telomerase activity in the cambium and adjacent zones of differentiating secondary xylem (Fig. 4).

Figure 4
figure 4

Anatomical observation of 5 years P. ginseng by tangential cryosectioning.

Aseries of 400-um-thick tangential cryosections (A) ~ (J) was taken for each sample: tissues at different stages were isolated by tangential cryosectioning; (B): Telomerase activities in various tissues and different stages of plant development were assayed by TRAP, using 47F as the forward primer and PTelC3 as reverse primers; (C): Densitometric quantization revealed higher relative telomerase activity (relative units) in cambiums.

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Analysis of TRF lengths in ginseng of different ages

DNA fragments were analyzed through DNA gel blot hybridization using the (CCCTAAA)3 oligonucleotide as a probe. A representative Southern blot image that was used to quantify TRFs is shown in Fig. 5a, b, where the hybridization signals represent telomeric regions. The autoradiograph was scanned and imported as a TIFF-format image to measure TRF length. The location of the peak intensity could not be accurately determined by eye. Therefore, an easy-to-use system that was able to determine the distributions of telomeric regions based on copy number and calculate statistics was employed. The unbiased TRF measure software Telotool25 was used to measure the TRF lengths of ginseng roots. A plot of the relative telomere copy number versus molecular weight was created, which provided the user with a realistic picture of the actual distribution of telomeric lengths. The measurements of TRF length for each sample using Southern hybridization was repeated three times.

Figure 5
figure 5

Southern hybridization images used for measurement and quantization of TRF length.

Lane M: DNA Molecular Weight Marker III, Digoxigenin-labeled (Roche). Numbers 2, 3, 4, 5, 6, 8 means different years of P. gensing samples collected from the city of Ji'an, Jilin province, China. B: Data fitting results and the trend of variation of TRF length with different ages. Overall, average TRF length increased with ages in main root (The following 1 cm of “ginseng lutou”).

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We investigated the correlation between TRF length and plant age using P. ginseng samples of known age from Ji'an and Fusong. First, DNA fragments were analyzed through Southern hybridization using the (CCCTAAA)3 oligonucleotide as a probe for telomeric DNA (Fig. 5a). Although observations made by eye are not precisely accurate, this easy-to-use method is convenient and allowed rapid analysis of the telomeres of ginseng roots by determining copy number26. A general model for age-related TRF length in ginseng was introduced. Eleven models were simulated using SPSS 20.0 software and the most suitable linear fitting curve was determined. The obtained results satisfied the 83% confidence limits (R2 = 0.832, F = 79.029, Sig. = 0.000) and it was found that TRF length was significantly positively correlated with age after 3 years (Fig. 5b), which indicated that TRF length could be maintained via telomerase activity as tissues developed. Based on these results, we propose a mathematical model through which telomere length can be used to predict P. ginseng age:

where, x is age and y is TRF length.

Discussion

Scientific identification of the potency of traditional Chinese medicines is crucial to ensure their authenticity and effectiveness. Authenticity can be assured based on several factors: the geographic origin or cultivation source of the species; proper harvesting and processing methods; and growth stage27. These factors are all important for the quality of Chinese herbal medicines. Because bioactive secondary compounds accumulate as medicinal plants such as P. ginseng, Salvia miltiorrhiza and Coptis chinensis age, older plants usually serve as better medicinal herbs. However, in the pursuit of economic efficiency, a number of inappropriate strategies, including the use of growth hormones and swelling agents as well as continual transplantation, have been employed to simulate age. Therefore, the quality of Chinese herbal medicines is difficult to determine. This study aimed to establish a reliable and effective method for identifying the age of ginseng that complements traditional methods of age determination.

Gymnosperms and dicotyledonous angiosperms generally undergo primary and secondary growth, whereas monocots usually lack secondary growth. The retention of stem-cell-like meristematic cells plays a critical role in perennial longevity28. Stem-cell-like meristematic cells are located in the cambium. Accordingly, when environmental conditions change periodically, associated with different growing seasons, the cambium cell cycle is activated and the tissue layers form rings (termed growth rings) during each individual period of growth. Arx, Schweingruber and Dietz29,30 indicated that growth rings could be an effective biomarker for estimating age in the roots of dicotyledonous perennial herbs. In the present study, growth-ring characteristics were clearly present in 1- to 6-year-old ginseng top roots. However, when the ginseng specimens were older than 6 years, dry, decayed channels emerged within the cambium rings, making the growth rings difficult to distinguish and influence age estimation. Therefore, this method can only be applied over a minimum age range and a new marker was required for estimation of the age of older ginseng samples.

Telomere length and telomerase activity are useful biomarkers for age prediction in animals and plants31,32,33 due to their close association with cell proliferation. However, it was unclear whether telomerase activity is related to the mechanisms maintaining stem cells in meristems. Our analyses of several P. ginseng tissues showed that telomerase activity was highest in the cambium. Telomerase expression in plants is very similar to that in humans. In plants, telomerase activity is highest in the meristem and reproductive organs, whereas there is little or no activity in the endosperm, leaves and stems17. In Ginkgo biloba, tissues with a high percentage of dividing cells also exhibit high levels of telomerase activity, which is consistent with our results31,34. We found that the sampled tissue had a substantial impact on the age estimation in P. ginseng. The main root samples contained most of the organized cambium and annual growth rings. We found that telomere length in the main roots was positively correlated with plant age. However, due to sampling limitations, ginseng plants of older ages were difficult to sample. Therefore, our mathematical model is only suitable for a certain range of ginseng ages.

A study that examined TRF branch length in detail suggested that telomere branch lengths increase with age to some extent in G. biloba31,35, in accord with the results of the present study. Our analyses indicate that ginseng telomere length increases significantly with age; however, in contrast to the progressive shortening of TRFs observed in somatic cells as animals aging, telomere length and telomerase activity change in different patterns during plant development. Telomere lengths have been observed to be stable in tomato leaves in four-week-old to six-month-old plants19 and they do not significantly change during plant ontogenesis or leaf senescence in Melandrium album18 and Arabidopsis thaliana36 or during cyclical changes of lengthening and shortening in size associated with age in Pinus longaeva20. Furthermore, telomeres do not shorten during increased tissue differentiation from embryonic to adult stages in Hordeum vulgare16 and Pinus sylvestris37, whereas they show decreased lengths during Betula pendula tissue culture38, while increased lengths are observed with age in G. biloba31. These results suggest that the relationship between telomere length and plant development is complex and may be affected by the species and lines involved as well as environmental stress and telomerase and stem cell activities. The present study indicates that telomere length in the top roots of P. ginseng increases with age, as observed in the leaves and calli of G. biloba31,35. Interestingly, many studies show that ginsenoside Rg1, which is one of the main biologically active components of ginseng, can decrease telomere shortening and reinforce telomerase activity in delayed hematopoietic stem cells and reduce senescence in human somatic cells39,40,41. Similar results were found for a G. biloba extract, which significantly augmented endothelial progenitor cell telomerase activity to prevent the cells from entering senescence42. These results imply that the increase in telomere length with age observed in ginseng and ginkgo may be related to the bioactive components of these plants, which may maintain telomere length by the telomerase mechanism or/and the ALT mechanism. The correlation between telomere length and telomerase activity in P. ginseng that was demonstrated here suggests that telomere length and telomerase activity might play essential roles in directly or indirectly regulating the life span of P. ginseng.

Methods

Sample collection

Ginseng samples of known age were collected from the Ji'an and Fusong districts of Jilin Province, China, in mid-August, 2010 and 2013 (Table 1). Samples of the main root (1 cm below the rhizome, known as “ginseng lutou” in China), leaf, stem, secondary root and seeds were frozen in liquid nitrogen and stored at −80°C until use.

Table 1 The Ginseng samples collected from two different districts
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Chromosome preparation and in situ hybridization

Adventitious roots was induced from calli of P. ginseng by cultured in the MS rooting medium for 14 d. Adventitious root tips were used as a source of metaphase chromosomes and the digoxigenin–dUTP nick (Roche, Penzberg, Germany) tag sequence (CCCTAAA)5 was used as a chromosome probe, as previously described43. Slides were removed immediately after immersion in 1 × PBS (containing 0.2% Tween) and dipped in 1 × blocking buffer (Boehringer, Ingelheim, Germany) at 37°C for 30 min. After drying, each slide was placed in 50 × blocking buffer containing 2 μl of a FITC-conjugated anti-digoxin antibody (anti-Dig FITC, Boehringer, Ingelheim, Germany), covered with a 22 × 22 mm coverslip and incubated in a dark, wet box at 37°C for 60 min. The slides were then washed three times (5 min each) in 1 × PBS (containing 0.2% Tween) at room temperature. After the slides were dried, 12 μl of the anti-fading agent VECTASHIELD® (Vector Lab, California, USA) containing 1 μg/ml propidium iodide (Life Technologies, California, USA) was added slowly to cover the coverslips. Different filters in a DMRXA fluorescence microscope (Leica Microsystems, Wetzlar, Germany) were then used to observe the red chromosomes and yellow-green hybridization signals. An air-cooled digital (CCD) camera was employed and the input of images into a computer was performed using Leica QFISH software to adjust the contrast and brightness.

Paraffin sectioning

Fresh roots of P. ginseng were harvested and samples were collected 1 cm from the root tip and fixed with FAA solution (70% 90:5:5 ethanol: formaldehyde: acetic acid), then vacuum infiltrated and dehydrated through increasing alcohol concentrations. Next, the sections were embedded in paraffin and the preparations were baked on an HI1220 flattening table and sectioned using an RM2265 rotary microtome (Leica Biosystems, Nussloch, Germany) to a thickness of 10–15 μm. The samples were subsequently baked for more than 24 h, then deparaffinized, stained with a safranin–fast green or phloroglucinol–HCl reagent, mounted with neutral gum and observed and photographed using BH-2 optical and LG-PS2 stereo microscopes (Olympus, Tokyo, Japan).

Hand sectioning

One-centimeter samples from the tips of fresh P. ginseng roots were sectioned at a thickness of approximately 1 mm. One drop of phloroglucinol-HCl was used to develop color and a scanner was employed to image the samples.

Tangential cryo-sectioning

A series of 40 μm-thick tangential sections were obtained for each sampled section as described by Uggla & Sundberg44, with some modifications. Regenerated tissues at different stages were isolated through tangential cryo-sectioning at −20°C with a Leica CM1850 Cryostat (Leica Biosystems, Nussloch, Germany). Cryosections of regenerated tissues from the same root and stage were collected in a 1.5 ml microfuge tubes, immediately frozen in liquid nitrogen and stored at −70°C.

Determination of telomerase activity

Total protein was harvested from approximately 5.0 mg of freshly ground, fine powder from each sample. Telomerase activity was measured as previously described45 using the TRAP assay. Plant extracts containing telomerase were prepared according to Fitzgerald17 and the total protein content in the extracts was determined46. The oligonucleotides 47F (5′-CGCGGTAGTGATGTGGTTGTGTT-3′) and PTelC3 (5′-CCCTAAACCCTAAACCCTAAA-3′) were used as forward and reverse primers, respectively, in the TRAP analysis18.

DNA extraction and Southern hybridization analysis

The TRF length, which is the gold standard for telomere length, was determined through Southern hybridization analysis47. Root samples were placed in a mortar and ground to a fine powder using a pestle and liquid nitrogen. Then, genomic DNA was prepared from each sample using the hot CTAB method48 and subsequently purified by treated with RNase (New England Biolabs, Massachusetts, USA) and Proteinase K (Merck, Darmstadt, Germany). The concentration of the isolated DNA and the ratio of the absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured using a NanoDrop ND-1000 spectrophotometer (Gene, Hong Kong, China). Approximately 20 μg of each DNA sample was then digested for 12 h with TaqaI and the digestion products were loaded into the lanes of a horizontal, 6.5 × 10 cm, 1% agarose gel and electrophoresed in 1 × TAE buffer for approximately 6 h at 90 V at room temperature with buffer recirculation. To measure and quantify the TRFs, Southern hybridizations were performed with the DIG High PrimeDNA Labeling and Detection Starter kit II (Roche, Penzberg, Germany) as previously described49,50 using an end digoxigenin-labeled complementary telomere-specific oligonucleotide probe (CCCTAAA)3. Measurements were repeated three times and TRF lengths are reported as the mean ± standard deviation.