ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
SEASON-SPECIFIC CHANGES IN TELOMERE LENGTH AND TELOMERASE ACTIVITY IN CHINESE PINE (Pinus tabulaeformis Carr.)1 © 2015 Y. Mu2, L. Ren2, X. Hu2, Y. Zhao, H. Li, H. Lu, D. Liu
College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing, P. R. China
Received December 12, 2014
Telomeres have lately received considerable attention in the development of deciduous tree species. In order to determine season-specific changes in telomere length and telomerase activity in evergreen tree species, Chinese pine trees (Pinus tabulaeformis Carr.) were used as experimental materials. In this study, we examined the correlation among telomere length, telomerase activity, and temperature in P. tabulaeformis during the course of an annual developmental cycle. A statistical analysis showed that the lengths of telomeres were significantly different between new and old leaves in each month. During the annual developmental cycle, the telomere lengths in Chinese pine tree leaves increased from May to June 2012, remained stable or increased slightly from June to August 2012, decreased sharply from August 2012 to January 2013, and then increased from January to April 2013. Telomerase activities could be detected in both new and old leaves from May 2012 to April 2013 and the telomerase activities in new leaves are higher than in the old each month. Additionally, there were similar trends between the changes in telomere length and mean monthly temperature from May 2012 to April 2013, and opposite trends were shown between the changes in telomerase activity and mean monthly temperature. Therefore, telomere length and telomerase activity varied with the season. Te-lomere length was positively correlated with temperature and telomerase activity was negatively correlated with temperature during the annual developmental cycle.
Keywords: Pinus tabulaeformis — season-specific — telomere length — telomerase activity — temperature
DOI: 10.7868/S0015330315040144
INTRODUCTION
The ends of linear eukaryotic chromosomes are protected by specific chromatin structures called telomeres that are composed of tandemly repeated telo-meric DNA and proteins [1]. Telomeres have important biological functions [2]. They protect chromosome ends and prevent the loss of terminal sequences during chromosome replication [1]. Telomeres in almost all higher plants are composed of the heptanucle-otide Arabidopsis-type telomere repeat (TTTAGGG)n [3, 4] with the exception of species in the order Aspar-agales and family Solanaceae [2]. Several families of the monocot order Asparagales contain the six base human-type telomere repeat (TTAGGG)n [4]. Another example of a non-canonical telomere repeat sequence
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations: TRFL — terminal restriction fragment length; TRAP — telomeric repeat amplification protocol; CTAB — hexa-decyl trimethyl ammonium bromide; CHAPS — 3-[(3-cholami-dopropyl)-dimethyl-ammonio]-1-propane sulfonate; BSA — albumin from bovine serum.
Corresponding author. Di Liu. College of Life Sciences and Biotechnology, Beijing Forestry University, Mail-box 162#, No. 35 Qinghua East Road, Haidian District, Beijing 100083, P. R. China; fax. +86-10-62338013; e-mail. liudi@bjfu.edu.cn
arose ~15 million years ago in several genera of the Solanaceae family [5]. The Arabidopsis-type telomere repeat is also present in gymnosperms [6].
Because of the inability of DNA polymerases to replicate linear DNA molecules completely, telomeres shorten with each round of DNA replication [7]. Telomeres are replicated by a specialized reverse transcriptase, called telomerase, by using their own RNA subunits as templates [8]. Telomerase activity was first found in Tetrahymena and then in a variety of different organisms from animal, yeast and plant [4].
Recent studies have shown that telomere length is species-specific. The telomeres in Arabidopsis thaliana ecotype Columbia span 2—5 kb [9], whereas tobacco (Nicotiana tabacum) telomeres are much longer, reaching 150 kb [10]. In contrast to members of the closely related Brassicaceae family, such as Arabidopsis thaliana, A. suecica, A. arenosa, A. lyrata, Capsella rubella, Olimarabidopsis pumila, and Brassica oleracea, telomeres in papaya (Carica papaya) are at least 10-fold longer and range in size from 25 kb to well over 50 kb [2]. In addition, telomere length depends on both the age of a cell and the number of times a cell has already divided [11]. Telomere lengths were found to change in a cyclical way by lengthening and shortening with age in Pinus longaeva needles [11] and Ginkgo biloba leaves [12]. Liu
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Fig. 1. Sampling of new and old needle leaves of Chinese pine (Pinus tabulaeformis Carr.).
Chinese pine is one of the evergreen gymnosperm species with needle leaves that grow well even in the autumn. According to the site of ovulate strobilus, we can determine the age of leaves. The new needles could not grow on the older branches. In our research, the age of new leaves were < 1 year (formed in spring 2012) and the age of old leaves was between 1 and 2 years (formed in spring 2011).
et al. [12] measured terminal restriction fragment lengths (TRFL) for different tissues from ginkgo trees and found that the rank order of telomere length in different tissues was leaf > callus > microspore. Aronen et al. [13] also found that different tissues of Scots pine (P. sylvestris) (immature embryos, cambium, buds, and needles of mature trees) had different telomere lengths. Changes of telomere length in ginkgo, ash (Fraxinus pennsylvanica var. subintegerrima) and willow (Salix matsudana) trees were shown to correlate with the season [14]. However, ginkgo, ash, and willow are all deciduous tree species whose leaves senesce and drop in the autumn. The very short telomere lengths in September and October in gingko, ash, and willow may be due to leaf cell apoptosis. Because all of the leaves are less than one-year-old, deciduous trees cannot fully reflect annual changes in telomere length corresponding to leaf age and temperature in trees.
Chinese pine (P. tabulaeformis) is one of the evergreen gymnosperm species with needles that grow well even in autumn and which usually live as long as four years. In addition, Chinese pine is a representative of Pinaceae species that is widely distributed in China, making experimental materials readily available. Chinese pine was used in this study to determine changes in telomere length and telomerase activity in the leaves and the correlation among telomere length, telomer-ase activity and temperature.
MATERIALS AND METHODS
Sample collection. Leaves from four Chinese pine trees (Pinus tabulaeformis Carr.) of the same age (45-year-old) were collected from free-growing trees on the campus of Beijing Forestry University (Beijing,
China, 40°00' N and 116°20' E). New leaves less than one-year-old leaves growing on current-year branches and old leaves (between one- and two-year-old) growing on two-year-old branches were collected (fig. 1) each month from May 2012 to April 2013. Temperature data for Beijing were obtained from the official website of the National Meteorological Center of CMA (http://cdc.cma.gov.cn/home.do). Each sample was frozen in liquid nitrogen and stored at —80°C.
Determination of telomere length. Samples were placed in a mortar and ground to a fine powder in liquid nitrogen using a pestle. The hot CTAB method [15] was used to extract sample DNA, which was quantified by spectrophotometry (NanoDrop 2000, "Thermo Scientific"). The integrity of the DNA samples was confirmed on an ethidium bromide-stained gel before blotting. TRF length was determined using Southern hybridization analysis as an indicator of telomere length [16]. DNA samples of approximately 20 ^g were digested for 36 h with HindYII. The digestion products were loaded onto a horizontal 6.5 cm x x 10 cm 0.8% agarose gel and electrophoresed in 1x TAE buffer for approximately 3 h at 100 V at room temperature. Southern hybridization was performed as described previously [12, 14] using a DIG High Prime DNA Labeling and Detection Starter Kit II ("Roche", Switzerland) with a digoxigenin end-labeled complementary telomere-specific oligonucleotide probe (5'-CCCTAAACCCTAAACCCTAAACCC- 3'). Telomere lengths were measured as described previously [12, 14].
Determination of telomerase activity. Telomerase activity was analyzed using the telomeric repeat amplification protocol (TRAP) [17]. The Coomassie protein determination method (with BSA reagent as the standard) was used to determine the protein content of the telomerase extract. PCR was performed as follows: initial de-naturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. An internal standard TSNT (5'-AATCCGTCGAGCA-GAGTTAAAAGGCCGAGAAGCGAT- 3') was used in PCR amplification, and CHAPS buffer was used as a negative control. The telomerase products were visualized by staining with ethidium bromide and photographed under UV light using an electronic gel documentation system [18]. Quantitation of telomerase activity was performed by densitometry [18—20].
Statistical analysis. The measurement of telomere length for each sample was repeated three times by Southern hybridization. Each telomere length is reported as the mean ± SD. The measurement of telom-erase activity for each sample was repeated three times by TRAP assay. Each telomerase activity level was reported as the mean ± SD. We analyzed the telomere lengths and telomerase activities from two aspects: (1) we compared telomere lengths and telomerase activities of the same month between new and old leaves from May 2012 to April 2013; (2) we measured telom-
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Fig. 2. Leaf telomere lengths in
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