Accepted Manuscript Cytohistological study of the leaf structures of Panax ginseng Meyer and Panax quinquefolius L Ok Ran Lee, Ngoc Quy Nguyen, Kwang Ho Lee, Young Chang Kim, Jiho Seo PII:
S1226-8453(16)30139-7
DOI:
10.1016/j.jgr.2016.08.001
Reference:
JGR 203
To appear in:
Journal of Ginseng Research
Received Date: 18 January 2016 Revised Date:
1 August 2016
Accepted Date: 2 August 2016
Please cite this article as: Lee OR, Nguyen NQ, Lee KH, Kim YC, Seo J, Cytohistological study of the leaf structures of Panax ginseng Meyer and Panax quinquefolius L, Journal of Ginseng Research (2016), doi: 10.1016/j.jgr.2016.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title
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Cytohistological study of the leaf structures of Panax ginseng Meyer and Panax
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quinquefolius L.
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Authors
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Ok Ran Lee1*†, Ngoc Quy Nguyen1†, Kwang Ho Lee2, Young Chang Kim3, Jiho Seo4
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Affiliations
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National University, Gwangju 61186, Korea
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Department of Plant Biotechnology, College of Agriculture and Life Science, Chonnam
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Science, Chonnam National University, Gwangju 61186, Korea
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Department of Herbal Crop Research, NIHHS, RDA, Eumseong 369-873, Korea
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Natural Resources Development Team, Ginseng Resources Research Institute, R&D
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Headquarters, Korea Ginseng Corp. Daejeon, Korea
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Department of Wood Science and Landscape Architecture, College of Agriculture and Life
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77 Yongbong-ro, Buk-gu, Gwangju 61186, Korea
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Department of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National
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University
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Corresponding author
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E-mail:
[email protected]
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Tel: +82 (0) 62 530 2054
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Fax: +82 (0) 62 530 2059
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†
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Running title: Leaf structures of shade-loving ginseng species
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These authors contributed equally to this work.
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ABSTRACT
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Background: Both Panax ginseng Meyer and Panax quinquefolius are obligate shade-loving
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plants whose natural habitats are broadleaved forests of Eastern Asia and North America. Panax
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species are easily damaged by photoinhibition when they are exposed to high temperatures or
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insufficient shade. In this study, a cytohistological study of the leaf structures of two of the most
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well-known Panax species was performed to better understand the physiological processes that
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limit photosynthesis.
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Methods: Leaves of ginseng plants grown in soil and hydroponic culture were sectioned for
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analysis. Leaf structures of both Panax species were observed using a light microscope, scanning
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electron microscope (SEM) and transmission electron microscope (TEM). Results: The
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mesostructure of both P. ginseng and P. quinquefolius frequently had one layer of non-
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cylindrical palisade cells and 3 to 4 layers of spongy parenchyma cells. P. quinquefolius
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contained similar number of stomata in the abaxial leaf surface but more tightly appressed
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enlarged grana stacks than P. ginseng. The adaxial surface of the epidermis in P. quinquefolius
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showed cuticle ridges with a pattern similar to that of P. ginseng.
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Conclusion: The anatomical leaf structure of both P. ginseng and P.quinquefolius shows that
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they are typical shade-loving sciophytes. Slight differences in chloroplast structure suggests that
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the two different species can be authenticated using TEM images, and light-resistant cultivar
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breeding can be performed via controlling photosynthesis efficiency.
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Keywords: Cytohistological study·Leaf·Panax ginseng·Panax quinquefolius·Thylakoid
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1. Introduction
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Ginseng (genus Panax; In Latin, the word panax means “cure-all”) is one of the most important
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perennial medicinal plants that belong to the family Araliaceae, and it has been cultivated for its
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highly valued medicinal properties for more than 2000 years in East Asian countries like China,
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Japan, and Korea [1, 2].
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The chromosome number of Korean ginseng (Panax ginseng Meyer) and American ginseng
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(Panax quinquefolius L.) has been reported to be 2n = 48 [3-5], and the plants are tetraploid (2n
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= 4x). Whereas some Panax species such as Panax notoginseng (Burk.) F. H. Chen have half
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number of chromosomes (2n = 24). P. ginseng has an estimated genome size of 3.12 Gbp per
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haploid chromosome equivalent [6], whereas P. quinquefolius has an estimated genome size of
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4.91 Gbp [7]. P. ginseng and P. quinquefolius (the closest relative of P. ginseng that recently
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diverged in the Panax lineage) [8] have been cultivated in shaded areas of Eastern Asia and
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forest canopies of eastern North America, respectively [9, 10]. However, information on the
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physiological differences between both ginseng species, especially cytohistological leaf
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structures that may affect photosynthetic activity, is not well characterized.
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Ginseng plants prefer north- and east-facing sites on well-drained slopes under a forest canopy
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with approximately 70%–90% shade. In Asia, ginseng plants have been traditionally cultivated
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under an impermeable straw hatch. In Korea, production was found to be higher when P. ginseng
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plants were grown in an experimental plot shaded with straw hatch than with a polyethylene net
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[11]. Several studies have tried to develop better shading materials for growing ginseng in Korea
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[12, 13]; China [14]; North America; and Ontario, Canada [15]. A physiological process that
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might limit photosynthesis was shown to be the result of smaller quantum yield of oxygen
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evolution in intact leaves of ginseng than in that of pea and spinach [16]. In this study, to
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investigate the differences between P. ginseng and P. quinquefolius, several morphological
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characteristics such as surface structure of the cuticle layer, number of stomata, and appressed vs.
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non-appressed thylakoids were observed.
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2. Materials and methods
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2.1. Plant materials and growth conditions Different aged P. ginseng cultivars ‘Chunpoong’, ‘Gumpoong’, ‘Yunpoong’, and ‘K-1’ and
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P. quinquefolius were kindly provided by the National Institute of Horticultural and Herbal
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Science (NIHHS) of the Rural Development Administration (RDA), Eumsung, Korea. Leaf
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tissue for stomata number counting was obtained from 3-week-old ginseng plantlets. In addition,
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the ginseng plants were hydroponically grown in perlite and peatmoss at 23°C ± 2°C under white
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fluorescent light (60–100 µmol m-2·s-1) in a controlled greenhouse. Ginseng cultivars grown in
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soil and under a polyethylene fabric cloth were obtained from a ginseng field (Suwon, Korea).
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2.2. Light microscopy and scanning electron microscopy
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For light microscopy, samples embedded in LR White resin (London Resin Co., London, UK)
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were thin-sectioned, stained with 0.1% toluidine blue, and examined using a light microscope
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(Zeiss, Axiolba, Germany). For scanning electron microscopy, the samples were fixed with a
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mixture of 2% glutaraldehyde and 2% paraformaldehyde and were post-fixed in 50 mM
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cacodylate buffer (pH 7.2) containing 1% osmium tetroxide (OsO4). After dehydrating the
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samples with a series of alcohols (30% for 40 min, 50% for 40 min, 70% for 40 min, 80% for 40
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min, 90% for 40 min, and 100% for 40 min twice), the samples were dried with a HCP-2 critical
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point dryer (Hitachi, Tokyo, Japan), coated with gold in an Emitec K550 ion sputter, and
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observed using a scanning electron microscope (SEM; Hitachi S-2400, Tokyo, Japan).
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2.3. Transmission electron microscopy Two-year-old ginseng leaf tips were fixed with a mixture of 2% glutaraldehyde (v/v) and 2%
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paraformaldehyde (v/v) in 0.05 M cacodylate buffer (pH 7.2) at room temperature for 4 h. The
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samples were washed using the same buffer and post-fixed with 1% OsO4 in 0.05 M cacodylate
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buffer at room temperature for 1 h. The fixed samples were washed with the buffer and then
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dehydrated in an ethanol series of 30%–100%. The samples were embedded in LR White resin
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at 50°C for 24 h, and ultra-thin sections (80–100 nm in thickness) were prepared using an ultra-
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microtome with a diamond knife. The thin sections were stained with uranyl acetate and lead
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citrate and then examined using a transmission electron microscope (TEM; JEM-1400, Jeol,
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Tokyo, Japan).
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3. Results and Discussion
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3.1. Original cultivation areas and characteristics of P. ginseng and P. quinquefolius
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USA and Canada, ranging from the southern part of Canada (Ontario and British Columbia) to
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central Alabama and from the east coast to the west of the Mississippi River, are the major
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producers of P. quinquefolius [17]. In South Korea, major P. ginseng cultivars have been
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cultivated in Daejon and Chuncheon between the latitudes of 36° and 38°N [13] (Fig. 1). A
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previous study showed that P. quinquefolius is more tolerant against greater light intensity than P.
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ginseng [18]; since plants that grow at higher latitudes receive greater light intensity than plants
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that grow at lower latitudes [14], this feature of P. quinquefolius seems possible. Cytohistological
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leaf structures of P. quinquefolius from Ontario and several P. ginseng cultivars [19, 20],
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Yunpoong, Chunpoong, Gumpoong, and K-1, were analyzed. Yunpoong is a high-yielding
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cultivar [21] with two to three more proliferated axillary shoots than the other P. ginseng
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cultivars [19]. Yunpoong has a relatively short stem length and a higher number of flower buds
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and seeds per plant [19]. Chunpoong is one of the best-fit cultivars for producing red-ginseng
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materials, with the longest main root length and an appropriate root diameter [19] for forming a
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human body shape. The lower half of the stem of Chunpoong is typically purple, and it has more
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curled leaves and exhibits a higher percentage of leaf burning than Yunpoong [19]. Disease
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resistance, especially against root rust, is the hallmark of the cultivar Gumpoong [21]. K-1 was
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recently registered as a high saponin producer and a high-yielding cultivar [21]. For detailed
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cytohistological observations on each ginseng species, P. ginseng cultivars and P. quinquefolius
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were grown in Suwon, Korea, under the same environmental conditions, and the leaf tips were
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analyzed (Fig. 2A).
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3.2. Abaxial and adaxial leaf-surface structures
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Both ginseng species showed similar adaxial leaf-surface structures (Fig. 2A, left), and they had
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the same patterns of cuticle ridges (Fig. 2B). Two different ginseng species also exhibited similar
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numbers of stomata located in the abaxial surface of the leaves grown in the same environmental
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condition (Fig. 2C). In a given same diameter (1.28 mm × 0.94 mm), 3-week-old P.
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quinquefolius with other analyzed P. ginseng cultivars did not show any significant difference on
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stomata number (Fig. 2C and 2D). A previous study showed that the density of stomata in young
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snake tomato leaves increased to 28% and 58% compared to the semi-old and old leaves [22].
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Evaluation whether ginseng leaf also show decreased number of stomata with an increase in age
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still in needs to be analyzed in near future. Sebastiania myrtilloides (Euphorbiaceae) is a good
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example of a plant that shows differential changes in leaf surface area between sunny and shady
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habitats; the number of stomata per leaf in the shady habitat was significantly lower than that in
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the sunny habitat [23]. The similarstructure of abaxial and adaxial leaf in P. quinquefolius and P.
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ginseng cultivars suggests that both species areevolved for shady habitat.
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3.3. Internal leaf structure with spherical palisade cells
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The internal space in leaves is composed of mesophyll cells, which are further divided into
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adaxial (upper) palisade and abaxial (lower) spongy cells in many dicotyledonous plants [24].
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The leaf skeleton of ginseng leaf shows reticulate venation with an elongated large vein, the
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midvein, per leaf. The midvein along the median longitudinal axis of the leaf in shaded field-
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grown 3-year-old P. ginseng cultivars and 4-year-old P. quinquefolius shows similar structural
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features with vascular bundle sheath cells in the center and collenchyma cells on both sides (Fig.
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3A). The leaves of the four selected P. ginseng cultivars and P. quinquefolius consisted of a layer
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of epidermis in the upper and lower sides; a layer of spherical palisade cells, which are distinct
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when compared with other sun-type plant species such as Capsicum annuum and Arabidopsis
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thaliana [26]; and two to three layers of spongy cells (Fig. 3A). The structural features were
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confirmed using hydroponically cultured 3-year-old P. ginseng cultivars and P. quinquefolius
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(Fig. 3B); this suggests that ginseng species of different ages have similar morphological internal
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leaf structures. One interesting aspect to be considered in ginseng leaf structure is that spherical
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palisade cells, rather than cylindrical palisade cells, are oriented perpendicular to the leaf surface
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[25]. Phototropins (PHOT1 and PHOT2) are blue-light receptors [26, 27]. PHOT2 mediates blue
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light-dependent development of cylindrical palisade cells [25]. When phot2 is mutated, spherical
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palisade cells, as observed in the ginseng leaves (Fig. 3), are formed. When plants are grown
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under high light conditions, leaves develop palisade tissues in which cylindrical cells are
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perpendicularly aligned to the epidermis to maximize the efficiency of photosynthesis [28-30].
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Further characterization of blue-light receptors in ginseng is required, especially to understand
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how ginseng species have evolved to adapt to the environment as shade-loving plants.
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3.4. Chloroplast and thylakoid structures
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Mesophyll cells of leaves have many chloroplasts, which contain the specialized light-absorbing
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green pigments, chlorophylls. Thus, the most active photosynthetic tissue in higher plants is the
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mesophyll in leaves. The most striking aspect of the structure of the chloroplast is the extensive
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system of internal membranes known as thylakoids. Chlorophyll is found within the thylakoid
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membrane system, which is the site of the light reaction of photosynthesis. TEM images showed
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that the ultrastructure of the chloroplasts in P. quinquefolius was ellipsoidal, whereas that of the
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chloroplasts in the P. ginseng cultivars was spherical (Fig. 4A). In barley, the shape of the
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chloroplasts is ellipsoidal in the light and an enlarged and round form in the dark [31]. Thus, P.
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quinquefolius used in this study may have originated from a higher-latitude area than the P.
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ginseng cultivars, and it has evolved for less densely shaded habitats. It cannot be excluded the
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possibility that P. quinquefolius used in this study might be acclimated to a given condition and
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may not represent its original habitat. Sun-type chloroplasts have more non-appressed thylakoids,
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and shade-type chloroplasts have more appressed thylakoids that comprise almost the entire
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chloroplast [32, 33]. Overall, the TEM ultrastructure images showed that both ginseng species
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were typical shade-adapted plants (Fig. 4). The chloroplast ultrastructure of the ginseng leaves
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showed large well-developed grana stacks that extended across the entire chloroplast (Fig. 4).
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The thylakoid membrane stacks of Yunpoong and Chunpoong were more loosely flattened than
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those of P. quinquefolius (Fig. 4B). The thylakoid structure of Gumpoong was more similar to
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that of P. quinquefolius (Fig. 4A) than that of Yunpoong and Chunpoong.
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4. Conclusions
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P. ginseng cultivars are derived from three lines, Jakyung, Chungkyung, and Hwangsook, by
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pure-line selection [20], and they are registered in the Korea Seed and Variety Service
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(http://www.seed.go.kr). Among nine cultivars, leaves of several superior P. ginseng cultivars,
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Yunpoong for a high-yielding variety from the Jakyung line, Chunpoong for red-ginseng
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manufacture from the Chungkyung line, and Gumpoong for disease resistance from the
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Hwangsook line were cytohistologically compared with the leaves of P. quinquefolius. As an
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obligate shade-loving plant, ginseng needs to be grown under an artificial cloth or under densely
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shaded forests. The most popularly used shade cloth in Korea is a lightweight knitted
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polyethylene fabric that protects plants from the sun. Thus, breeding ginseng cultivars to be
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grown under natural sunshine without any artificial shading will greatly reduce total cultivation
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expenses. Crossing two different ginseng species can result in more light-resistant cultivars. P.
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quinquefolius generally shows a more light-adapted phenotype than several P. ginseng cultivars.
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The anticlinal walls of the adaxial surface of leaf epidermal cells were wavy and showed similar
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patterned cuticle ridges in P. quinquefolius and P. ginseng cultivars (Fig. 2B). The microscopic
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characteristics of the surface view were the same as those of a previously characterized 5-year-
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old P. quinquefolius plant [34]. The abaxial surface of the leaf epidermis was also wavy in the
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two different ginseng species with similar number of stomata (Fig. 2C and D). The density of
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stomata in leaves in a sunny habitat is significantly higher than that in leaves in a shady habitat
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[23]. Highly dividing cells from leaf tips showed similarly structured forms in the midvein, both
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sides of epidermis cells in the midvein, and vascular bundles (Fig. 3A). The most striking
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features in the mesostructure of the ginseng leaves were the one layer of palisade cells, which are
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commonly cylindrical but were rather spherical and oval (Fig. 3B). The leaf mesophyll consisted
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of 3 to 4 cell layers of spongy parenchyma cells (Fig. 3) in P. quinquefolius and P. ginseng
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cultivars. Overall surface and internal leaf structures suggest that P. ginseng and P. quinquefolius
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are both sciophytes with few cells and chloroplasts per unit leaf area. This finding corresponds
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with that of a previous study on P. quinquefolius grown in Russia [35]. An important feature of
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shade plants is their chloroplast size and ultrastructure. The density of grana stacks in shade
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plants is always higher than the density of those in chloroplasts from plants exposed to full
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sunlight [32, 33, 36, 37]. The shape of chloroplasts from P. ginseng was rounder than the
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ellipsoidal chloroplasts from P. quinquefolius (Fig. 4). A previous study showed that light-
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illuminated chloroplasts are ellipsoidal rather than round and enlarged during the dark period
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[31]. P. quinquefolius seemed more adapted to sunny conditions than the P. ginseng cultivars.
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The thylakoid membranes in P. quinquefolius were also appressed tightly with distinctly apparent
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grana stacks and decreased stroma volume. However, the most popular P. ginseng cultivars,
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Yunpoong and Chunpoong, showed relatively loosely stacked grana extended over the entire
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chloroplast body (Fig. 4B). Thus, the two different ginseng species are shade-loving sciophytic
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plants that share most ultrastructural features such as stacks of grana and stroma. The results
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obtained in this study can be useful not only for breeding light-adaptable cultivars but also for
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authentication of different widely cultivated species.
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Conflicts of interest
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The authors declare that there are no conflicts of interest.
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This study was financially supported by a grant from the Next-Generation BioGreen 21 Program
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(SSAC, grant: PJ01112901); Rural Development Administration, Republic of Korea.
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Figure legends
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Fig. 1. Major Panax ginseng and Panax quinquefolius cultivation areas located worldwide.
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tips used for surface structure analysis (left). Diagram of 3-week-old ginseng plantlet (right)
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Five-leaf structure is additionally emerged as per age with no morphological changing in other
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organs. (B) SEM image showing the adaxial (upper) surface of a ginseng leaf with wavy
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anticlinal cuticle ridges. Bars = 20 µm (left) and 10 µm (right). (C) In a diameter (1.28 mm ×
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0.94 mm) of 3-week-old ginseng leaves, the total number of stomata is similar among Panax
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quinquefolius and three Panax ginseng cultivars in 1st and 2nd leaves depicted in Fig. 2A (right).
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Randomly selected 8 different diameter images were used to count (n=8) (D) SEM image
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showing the abaxial (lower) surface of a ginseng leaf with wavy epidermis and similar numbers
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of stomata. Bars = 20 µm (left) and 10 µm (right).
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Fig. 3. Internal leaf structures of ginseng. (A) Midvein, leaf (circled uppermost part of leaf from
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Fig. 2A), adaxial and abaxial side of the midvein, and vascular bundle structure of soil-grown
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Panax ginseng cultivars and Panax quinquefolius observed using light microscopy. Bars = 5 µm
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(the uppermost row) and 20 µm. (B) Mesostructures of ginseng leaves from 3-year-old
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hydroponically cultured ginseng species. Bars = 50 µm
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Fig. 4. Chloroplast and magnified thylakoid membrane structures. (A) Chloroplast morphology
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of three Panax ginseng cultivars and Panax quinquefolius is different. Bars = 5 µm. (B)
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Magnified chloroplast structure shows differently stacked thylakoid membranes. Bars = 500 nm.
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Fig. 1.
A
B
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Yunpoong
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The number of stomata on abaxial leaf (n)
American ginseng
P. ginseng cultivars
Fig. 2.
Yunpoong
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American ginseng
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Gumpoong
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Adaxial side of midvein
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Fig. 3.
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Yunpoong
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Fig. 4.
P. quinqueforius
