Anatomical Development of Cell Structure Including Casparian Strip During Root Growth in Grapevines

Anatomical Development of Cell Structure Including Casparian Strip during Root Growth in Grapevines

Yang Song, Lihong Ye, Astha Tuladhar and Naosuke Nii*

Faculty of Agriculture, Meijo University, Tempaku, Nagoya 468-8502, Japan

Morphological and anatomical changes in roots associated with their development were investigated in grapevines. When new roots initiated from the suberized structural root in grapevines, the tip of the root expanded outward and the suberized outer layer was detached from the surface of the root tip. At the onset of root initiation, the cell nucleus reappeared in the apical meristem, and cell division occurred. Once approximately five xylem vessels per pole had appeared, the Casparian strip appeared as a dot in the center of the radial wall of the endodermis. At a point 150 mm from the root tip, the endodermis was completely suberized and the number of xylem vessels per pole increased. In younger roots, suberization of the exodermis was observed earlier than in the endodermis, although the Casparian strip could not clearly be detected in the exodermis. As the root aged, a gap formed between the endodermis and pericycle. Before the endodermis was sloughed off from pericycle layers, cell division was observed in the pericycle and the suberized pericycle layers increased.

Key Words: endodermis, exodermis, pericycle, root growth, suberization.

growth in relation to water and nutrient movement. Introduction During root aging, the shedding of cortical tissue from The roots and root systems of the grapevine Vitis spp. growing roots is a natural process in all woody perennial have been reviewed in detail by Richards (1983). This plants, including grapevines. When the endodermis has review placed particular emphasis on the anatomy and separated from the pericycle layer during the root- morphology of grapevine roots in relation to the soil growing period, the number of pericycle layers and the environment, and also described root function and the extent of suberization have not been fully investigated. interrelationship between the root and shoot systems; In the present report, we followed the root terminology however, several issues need to be resolved in order to of Esau (1977) and McKenry (1984). better establish the anatomical characteristics during the The present investigation focused on the anatomy and root growth of grapevines. Firstly, root initiation from morphology of grape roots in relation to root function, the tips of structural roots and their subsequent with particular emphasis on the following: (1) root elongation occurs in perennial fruit trees, such as initiation from structural roots and (2) root growth in grapevines, as a means of increasing root number and relation to Casparian strip formation and suberization in the extent of the root zone. Detailed knowledge of the the exodermis and endodermis, and separation of the changes in root structure is very important for endodermis and pericycle including periderm formation. understanding root growth from the structural roots. Materials and Methods Secondly, although the differentiation and development of the Casparian strip has been already reported for Anatomical and morphological changes in roots during several fruit trees, e.g. pear (Esau, 1943) and apple root initiation from structural roots (MacKenzie, 1979; Riedhart and Guard, 1957), the The characteristics of root initiation from structural relationship between the Casparian strip and root roots were investigated in two-year-old cuttings of development should be also investigated in order to ‘Delaware’ (Vitis labruscana Bailey) grown outdoors in understand the mechanisms employed by grapevine 500 mL pots filled with sandy soil. In April, root samples of different ages were selected by judging the color of Received; June 25, 2010. Accepted; December 4, 2010. root tips; new roots were white and structural roots were * Corresponding author (E-mail: [email protected]). brown.

164 J. Japan. Soc. Hort. Sci. 80 (2): 164–168. 2011. 165

Root tip morphology was firstly observed under a dissecting microscope (SZX12, Olympus, Japan). The root samples were then fixed in 3% glutaraldehyde (0.1 M cacodylate buffer, pH 7.2) and stored at 4°C. The samples were then dehydrated through a graded ethanol series and embedded in Technovit 7100 (Kulzer, Germany). Semi-ultrasections (1.5 μm) cut by a glass knife were stained using methylene blue for histological examination under a light microscope (BX60, Olympus).

Anatomical analysis of the roots, including Casparian strip formation, suberization of exodermis and endodermis, and periderm formation Anatomical development during root growth was investigated in two-year-old cuttings of ‘Muscat Bailey A’ (V.vinifera × V.labruscana) grown in a greenhouse in 500 mL pots filled with sandy soil. Roots samples were collected in September. The external appearance of the roots used in the present study was creamy white up to 150 mm from the root tip, after which roots turned a light brown to the basal portion of the root. Transverse sections, commencing approximately 5 mm from the root tip and then every 10 mm to the root base, were cut by hand to examine the extent of structural development in relation to root age. Detection of autofluorescence (excitation wavelength: 365 nm) of the Casparian strip and suberin lamellae was achieved by examining unstained root sections under UV light (BX60, Olympus). Occasionally, although the figures did not give an example, Sudan red 7B (Sigma, USA) Fig. 1. Appearance of grape roots. A, root of two-year-old grape or berberine (Sigma) was selected for better confirmation cutting of ‘Delaware’. Large arrows point to new roots and small of Casparian bands (Brundrett et al., 1988). For arrow points to new lateral root; B, structural root; C, root examining the anatomical features of the cells, the initiation; D and E, new roots from the suberized structural root. = remaining root samples were prepared for sectioning by Scale bars 1 mm (Panels B–E) and 10 mm (Panel A). the Technovit 7100 embedding method and observed by methylene blue staining. Sections were examined by a from their suberized tips, even after long periods. In the light or fluorescent microscope as described above. present study, extensive cell division was observed in the apical meristem zone when roots restarted to elongate Results and Discussion and a large nucleus appeared in these cells (Fig. 2D, F). Anatomical and morphological changes in roots during Although the nuclei in the apical meristems of structural root initiation from structural roots roots were small and few in number, nuclei in the cells Although new lateral roots were also observed, of the apical meristem at the beginning of root initiation extensive root initiation occurred from the tips of the were typically well defined and large; thereafter, new structural roots (Fig. 1A). The color and shape of the roots typically elongated due to cell extension. tips of structural roots and root initiation were apparently distinguishable (Fig. 1B, C); structural roots were brown Anatomical analysis of the roots including Casparian and dome-shaped, while initiation roots were white and strip formation, suberization of exodermis and pointed. Cell nuclei were not observed in sections of endodermis, and periderm formation meristematic areas in structural roots (Fig. 2A, B). The Transverse sections of the roots were observed from extent of cell division in the apical meristematic zones the tip towards the base of the root (Figs. 3 and 4). At of structural roots appeared to be low. After the initiation 10–50 mm from the root tip, the vascular bundle, of new roots from the tip of the suberized structural root, particularly the xylem vessels, was not clearly observed the upper portion of the roots swelled outward and the (Fig. 3B, D). Specifically, there was only one xylem suberized outer layer was detached from the surface of vessel per pole and several phloem cells. At 100 mm the root tip (Figs. 1D, E and 2C, E). As reported from the root tip, the secondary xylem vessels were previously by Pratt (1974), completely suberized grape clearly apparent and more elements of the phloem had roots in dry soil could resume growth by regeneration developed (Fig. 4B, C). The endodermis could be clearly 166 Y. Song, L. Ye, A. Tuladhar and N. Nii

Fig. 2. Photomicrographs of longitudinal sections of grape root tips of ‘Delaware’ after staining with methylene blue, showing anatomical differences of apical meristem. Root samples were embedded in Technovit 7100 and sections (1.5 µm) were cut with a glass knife. A, structural root; C and E, root initiation; B, D, and F, enlarged photos of root apical meristem in A, C, and E, respectively. Large nucleus appeared in new roots (arrows Fig. 4. Transverse sections at each point from the root tip to the basal point to nucleus). Scale bars = 100 µm (Panels B, D, F) and portion in ‘Muscat Bailey A’. Root samples and sections were 500 µm (Panels A, C, E). prepared using a similar method to Figure 3. A, 100 mm from the root tip; E, 180 mm from the root tip; G, 510 mm from the root tip; B, C, and D, enlarged photo of endodermis, pericycle and vascular bundle in A; F and H, enlarged photos of pericycle in E and G, respectively. Large arrows indicate pericycle division (B, D), small arrows indicate secondary xylem vessel (B) and arrowheads indicate cork cambium (phellogen) (F, H). Scale bars = 50 µm (Panels C, D, F, H), 100 µm (Panel B), and 200 µm (Panels A, E, G).

observed 10 mm from the root tip. At a younger stage of root growth, many particles in the endodermis were observed, such as lipids and/or phenolic compounds (Fig. 3B, D). Previous studies have shown that such lipid deposition in the endodermis occurred before complete suberin lamella formation (Barnabas and Peterson, 1992; MacKenzie, 1983). These compounds diffused through- out the endodermis cells 60 mm from the tip (Fig. 3F). Van Fleet (1961) demonstrated that the endodermis can be histochemically differentiated at a very early stage by its high phenol content. At 100 mm from the root tip, pericycle division occurred (Fig. 4B, D) and some of the Fig. 3. Transverse sections at each point from the root tip to the basal portion in ‘Muscat Bailey A’. Root samples were embedded in accumulated compounds in the endodermis reduced Technovit 7100 and sections (1.5 µm) were stained with (Fig. 4C–F). methylene blue for light microscopy. A and B, 10 mm from the At 10 mm from the young root tip, the xylem and root tip; C and D, 50 mm from the root tip; E and F, 60 mm phloem components of the vascular bundle become from the root tip; A, C, and E, outer portions of the root; B, D, alternately positioned (Fig. 3A, B). At approx. 70– and F, inner portion including endodermis and vascular bundle. Large arrows indicate endodermis (B, D, F) and small arrows 100 mm from the root tip, the secondary xylem vessels indicate xylem vessel (B, D). ph, phloem (B, D, F). Scale bars appeared on the inner side of the cambium (Fig. 4A, B). = 50 µm (Panels B, D, F), 100 µm (Panels C, E), and 200 µm Later, the xylem developed under the phloem due to (Panel A). cambium division. The number of xylem vessels per pole was observed to change with increasing distance from the root tip or with root age. Consequently, J. Japan. Soc. Hort. Sci. 80 (2): 164–168. 2011. 167 assessing the number of xylem vessels per pole appears poles. In the present study, suberization in the to be well suited for estimating root age in grapevines. endodermis with the basal portion was observed to differ Suberized exodermis was observed in younger roots between cell/tissue types, with some cells opposite the (Fig. 5A, C, E). The localization of suberin in the xylem poles remaining unsuberized (Fig. 5F). Suberiza- exodermis enables protection of the tissues against tion of the exodermis was observed earlier than the desiccation in the middle of the cortex. The exodermis, endodermis in grape roots. as with the endodermis, also develops the characteristics Brown patches of reduced root diameter were of an apoplastic barrier. In the fibrous roots of grapevines, occasionally observed along the length of white roots the epidermis is a transient cell layer and the hypodermis due to the collapse of cortical tissue (Fig. 6A, B). The develops lignosuberized Casparian bands (Perumalla et reduction in the diameters of the main roots coincided al., 1990); however, in the present study, the Casparian with the separation of the endodermis from the pericycle strip could not clearly be detected in the exodermis. (Figs. 4G, H and 6). When the cortical tissue with the Accompanying xylem development, the Casparian endodermis detached from the pricycle, the number of strip in the endodermis was observed to expand in the pericycle layers increased and accumulated auto- entire radial wall (Fig. 5). At 10 mm from the root tip, fluorescent compounds (Fig. 6C, D). The pericycle, the Casparian strip initially only appeared as a dot in which was initially uniseriate, became multiseriate by the middle of the radial wall of the endodermal cells tangential divisions (Fig. 4D). The beginning of pericycle (Fig. 5B, D). The extension of suberization with the division corresponded to the development of secondary Casparian strip in the endodermis coincided with the xylem vessels (Fig. 4B). The outermost cells resulting root’s age/or with an increase in xylem vessel number. from these divisions became cork cells (Fig. 6B–D), and MacKenzie (1979) demonstrated that Casparian strips beneath these cells arose a cork cambium (Fig. 4F, H). differentiated at the same time in all endodermal cells Before separation, at 180 mm from the root tip, the in apple roots. Reinhardt and Rost (1995) described that number/thickness of pericycle layers typically increased Casparian bands in the endodermis in cotton roots and the cortex adjacent to the endodermis was crushed appeared synchronously in the radial walls. In addition, by expansion of the vascular bundle (Fig. 4E, F). passage cells without suberin lamellae were usually Separation of the endodermis from the pericycle present in the endodermal layer opposite protoxylem appeared to occur after suberization with the Casparian strip within endodermis cells (Fig. 6C, D). During this period, phloem fibers began to lignify (Fig. 6B) and cork cells sloughed off continuously one after another from the outside during root growth (Fig. 6C, D) as reported in roots of the grapevine and apple (Richards, 1983; Riedhart and Guard, 1957).

Fig. 5. Hand cross-sections of grape root showing development of the Casparian strip and suberization in the endodermis and exodermis in relation to vascular bundle development in ‘Muscat Fig. 6. Photomicrographs of cross-sections of grape root showing Bailey A’. Root sections were examined by fluorescent formation of the periderm and separation from the pericycle in microscopy without staining. A, 10 mm from the root tip; C, ‘Muscat Bailey A’. Root samples were embedded in Technovit 70 mm from the root tip; E, 150 mm from the root tip; B, D, 7100. Sections (1.5 µm) were stained with methylene blue and and F, enlarged photos of endodermis and vascular bundle in examined by light microscopy (A, B) or fluorescent microscopy A, C, and E, respectively. Large arrows indicate the exodermis (C, D). A and B, periderm formation; C and D, separation and and small arrows indicate Casparian strips in the endodermis; shedding from pericycle. Small arrow indicates phloem fiber Arrowheads, passage cells (F). Scale bars = 100 µm (Panels B, (B). CO, cork cell (B–D). Scale bars = 100 µm (Panels C, D), D, F) and 200 µm (Panels A, C, E). 200 µm (Panel B), and 500 µm (Panel A). 168 Y. Song, L. Ye, A. Tuladhar and N. Nii

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