ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 3, с. 389-394
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
y%K 581.1
SPATIAL ACCUMULATION OF CAMPTOTHECIN ASSOCIATED WITH ARBUSCULAR MYCORRHIZA DEVELOPMENT IN ROOTS
OF Camptotheca acuminate SEEDLINGS1 © 2014 Y. Wu***2, Y. Yu****2, G. E. Xie*****2, Q. Y. Pang*, Y. Wang*, X. F. Yan*
*Alkali Soil Natural Environmental Science Center, Northeast Forestry University/Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, China **College of Life Sciences, Northeast Forestry University, Harbin, China ***Key Laboratory for Molecular Enzymology & Engineering (Jilin University), Ministry of Education, Changchun, China
****Benxi Chemical Industry School, Benxi, China Received March 20, 2013
Camptotheca acuminate plant is an important phytomedicinal species that contains camptothecin (CPT), a natural pentacyclic indole alkaloid. This study was aimed at the effect of arbuscular mycorrhiza (AM) development on the accumulation and location of this metabolite in C. acuminate seedling roots. Autofluorescence of CPT shows the localization of CPT in mycorrhizal roots. We examined CPT level in response to AM development in C. acuminate roots by the combination of confocal laser scanning microscopy and spontaneous fluorescence of CPT. CPT was specifically localized around AM fungi in AM roots of C. acuminate, and the level of CPT rose with increasing colonization of roots with AM fungi. The accumulation and localization of CPT was directly correlated with the formation and development of AM in C. acuminate roots.
Keywords: Camptotheca acuminate - arbuscular mycorrhiza - camptothecin - autofluorescence - confocal microscopy
DOI: 10.7868/S0015330314030142
INTRODUCTION
Arbuscular mycorrhiza (AM) is the most widespread symbiosis formed between roots of 70-90% of land plant species and fungi of the order Glomales [1]. AM improves plant nutrient uptake from the soil and receive photosynthetic carbohydrates in return [1]. This intimate interaction is associated with drastic changes in the physiology and ecology of the plant. AM colonization enhances plant growth and improves plant resistance against pathogen, heavy metal, and salinity [1].
Secondary metabolites play multiple functional roles in plant response to various abiotic and biotic environments, and increasing evidence indicates that they may be closely related to mycorrhizal symbioses [1]. For example, during the establishment of AM symbiosis, the levels of flavonoid/isoflavonoid com-
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations: AM - arbuscular mycorrhiza; CLSM - confocal laser scanning microscopy; CPT - camptothecin. Corresponding author: Xiufeng Yan. Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Harbin 150040, China. Fax: +86 (451) 82-19-0052. E-mail: xfyan@ nefu.edu.cn
pounds altered in mycorrhizal roots of many plants, such as Medicago sativa, M. truncatula, and soybean (Glycine max) [2, 3]. Moreover, cyclohexenone gluco-sides are exclusively synthesized in the roots colonized by AM fungi [4, 5]. The concentration of trigonelline increased in AM roots of Prosopis laevigata [6]. However, little is known about the relationship between spatial accumulation of secondary metabolites and AM development in this symbiosis system.
Camptothecin (CPT), a monoterpenoid indole alkaloid produced by Chinese tree Camptotheca acuminate (Nyssaceae) exhibits potent anti-tumor properties [7]. For the pharmaceutical importance of CPT and C. acuminata, the variation of CPT in C. acuminate seedlings and the effect of various stresses on the CPT content have been documented [7]. We have found that AM fungi could significantly improve the C. acuminate seedling growth and the CPT concentration in roots [8]. Here we report the temporal and spatial variation of CPT coupling with the development of the AM fungi in C. acuminate seedlings at cellular level by using the combination of confocal laser scanning microscopy (CLSM) and spontaneous fluorescence of CPT
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MATERIALS AND METHODS
Cultivation of mycorrhizal plants. Seeds of Camp-totheca acuminate (Jintang, Sichuan, China) were sterilized with 0.5 g/mL KMnO4 for 0.5 h and washed with sterile water. They were imbibed overnight in sterile water and transferred to sterile plates containing sterilized sand moistened with water to germinate. After the first couple of true leaves has developed, each seedling was planted into sterilized pot (20 cm in diameter, 20 cm in depth) with inoculated substrate. The inoculated substrate consisted of steam-sterilized (120 min, 121°C) mixture of sand and clay (1 : 3 by volume, 2 mm-sieve) mixed with 30 g of soil (control treatment) or 30 g of soil containing Acaulospora mel-lea Spain & Schenck spores. Plants were grown in chambers at 23-25°C, 70% relative humidity, and a light/dark regime of 16/8 h.
Estimation of root colonization. Roots were collected every 5 days after 7-day-long inoculation, carefully washed, and cleared in 100 g/L KOH for 50 min at 90°C. After careful washing, the roots were stained with Trypan blue for 20 min at 90°C, and then placed in discoloring solution (lactic acid/glycerol 1 : 1, v/v), in order to visualize the fungal structures inside the roots (a modified method of Phillips and Hayman [9]). Mycorrhizal parameters, including the frequency of mycorrhiza in the root system (F, %), the intensity of the mycorrhizal colonization in the root system (M, %), the intensity of the mycorrhizal colonization in the root fragments (m, %), arbuscule abundance in mycorrhizal parts of root fragments (a, %), and arbuscule abundance in the root system (A, %), were calculated according to Trouvelot method [10] by the following formulae:
F = (nb of fragments myco/total nb) x 100, where nb means the number;
M = (95n5 + 70n4 + 30n3 + 5n2 + n1)/total nb, where n5 - the number of fragments rated 5; n4 - the number of fragments 4, etc.;
m = M x total nb/nb of fragments myco;
a = (100mA3 + 50mA2 + 10mA1)/100, where mA3, m^, mA1 are the % of m, rated A3, A2, A1, respectively, with mA3 = ((95n5A3 + 70n4A3 + 30n3A3 + 5n2A3 + n1A3)/nb of fragments myco) x 100/m, where n5A3 -the number of arbuscule abundance rated 3 in n5, n4A3 -the number of arbuscule abundance rated 3 in n4, etc., and the same for m^ and mA1;
A = a x M/100.
The experiment was performed three times with three replicates.
CLSM analysis of AM structures and cellular localization of CPT. Localization of camptothecin in mycorrhizal roots was investigated by detection of the spontaneous fluorescence of CPT with CLSM. Root segments were cut to approximately 1-cm segments, embedded in glycerol, and frozen in embedding com-
pound (OCT compound, "Sakura Finetek", United States). 60-^m-thick longitudinal sections were cut with a Leica cryotome (CM 1900, "Leica", Germany). Sections were stained overnight in a solution containing lactic acid, glycerol, distilled water, and 0.01 g/mL of acidic fuchsin [11]. Sections were mounted in 15% glycerol between glass spacers on glass slides, covered with a cover slip, and sealed with nail polish. Autofluorescence microscopy of CPT was performed by CLSM (LSM 510 Meta; "Zeiss", Jena, Germany) and filter set with excitation at 364 nm and emission at 435-485 nm according to the method modified of Pasqua et al. [12] and Sirikantaramas et al. [13]. Excitation at 488 nm was used to obtain the bright-field image of AM-colonized roots sections.
RESULTS
AM morphology and intensity of mycorrhizal
colonization
A. mellea strains successfully colonized C. acuminate roots and formed Arum-type colonization with appressoria, intracellular hyphae, intracellular arbus-cules, and vesicles (figs. 1a-1c). The vesicles and ar-buscules were observed in the cortex of the main and lateral roots.
In order to assess the effect of AM development on CPT accumulation in the root of C. acuminate seedling, the frequency (F, %), intensity (M, %), and arbuscule abundance (A, %) of AM fungal root colonization were calculated every 5 days after 7-day-long inoculation. As shown in fig. 1d, the colonization was low during 17 initial days of observation after inoculation and increased significantly thereafter. The most effective colonization (F = 92.2%, M = 68.5%, A = 13.2%) was detected after 40 days of observation, and most of the fungal structures were present, for example, hyphae, arbuscules, and vesicles. At day 32, the lower colonization (F = 44.4%, M = 13.7%, A = 0.30%) was observed. This decreased colonization might associate with the degradation of early colonization units. There was no colonization in the non-AM control plants.
Accumulation of CPT upon fungal root colonization
Autofluorescence of CPT provides a direct way to investigate the localization of CPT in mycorrhizal roots of C. acuminata. By comparing the intensity of fluorescence, relative concentrations of CPT in various stages of the AM symbiosis can be examined. The staining procedure of AM root sections was a prerequisite to obtain bright-field image of highly mycor-rhizal structures. Autofluorescence of AM structures was observed under blue light excitation, but not under UV or green light [14], which did not interfere with CLSM analysis of CPT detection under UV light. Typical CPT accumulation pattern in A. mellea-colo-nized C. acuminate roots is presented in fig. 2. We ob-
Days of colonization
Fig. 1. AM morphology and mycorrhizal colonization of Camptotheca acuminate roots treated with Acaulospora mellea inoculation. a—c — typical Arum-type morphological structures: external hyphae (eh), appressoria (ap), intercellular hyphae (ih), arbuscule (ar), and vesicle (v), shown by Trypan blue staining; scale bar — 20 ^m; d — frequency of mycorrhiza (F, %) (1), mycorrhizal intensity (M, %) (2), and arbuscule abundance (A, %) (3) of C. acuminate roots treated with A. mellea fun
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