Anatomical and histochemical features of Brasenia schreberi (Cabombaceae) shoots

Journal Pre-proof Anatomical and histochemical features of Brasenia schreberi (Cabombaceae) shoots Chaodong Yang, Xia Zhang, James L. Seago Jr, Qingfeng Wang

PII:

S0367-2530(19)30528-6

DOI:

https://doi.org/10.1016/j.flora.2019.151524

Reference:

FLORA 151524

To appear in:

Flora

Received Date:

9 February 2019

Revised Date:

31 October 2019

Accepted Date:

27 November 2019

Please cite this article as: Yang C, Zhang X, Seago JL, Wang Q, Anatomical and histochemical features of Brasenia schreberi (Cabombaceae) shoots, Flora (2019), doi: https://doi.org/10.1016/j.flora.2019.151524

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Title: Anatomical and histochemical features of Brasenia schreberi (Cabombaceae) shoots

Running head: Anatomy for Brasenia schreberi shoots

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Authors: Chaodong Yang 1, Xia Zhang1, James L. Seago Jr2, Qingfeng Wang3*

Engineering Research Center of Ecology and Agriculture Use of Wetland, Ministry

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of Education, Yangtze University, Jingzhou, 434025, Hubei, China

Department of Biological Sciences, State University of New York, Oswego, NY

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13126, USA

Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical

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Garden, The Chinese Academy of Sciences, Wuhan, Hubei,430074, China

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Correspondence authors e-mail: Qingfeng Wang, [email protected]

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Co-Authors e-mail: Chaodong Yang, [email protected]; Xia Zhang, [email protected];

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James L. Seago Jr., [email protected]

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Highlights

The shoots of B. schreberi have vertical and horizontal rhizomes, and stolons with peduncles and leaves. The stems have polysteles with bicollateral bundles, endodermis, aerenchyma and discontinuous cuticles. Glandular trichomes compose of two disk-like stalk cells, and enlarged glandular cells with storage space and mucilage. 1

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These structural features of B. schreberi adapt to an aquatic environment.

Abstract: The present research on Brasenia schreberi, a member of the aquatic, basal angiosperm family, Cabombaceae, investigates the anatomy and histochemistry of this critically endangered plant. The shoots of B. schreberi have vertical rhizomes, horizontal rhizomes, and stolons with peduncles and leaves. Most stems typically have polysteles with bicollateral vascular bundles, each with two groups of primary

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xylem tracheids and primary phloem sieve tube elements, an endodermis with Casparian bands around each vascular bundle, and epidermis with glandular

trichomes or “mucilaginous hairs” and discontinuous cuticle. Most stems have a

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protoxylem lacuna between the two regions of primary xylem and primary phloem in each bicollateral vascular bundle and extensive aerenchyma in ground tissues.

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Epidermal glandular trichomes are composed of two disk-like stalk cells and enlarged glandular or hair cells with storage space and mucilage, which covers the cells. The

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structural features of B. schreberi are generally consistent with those of other aquatic

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basal angiosperms and with its adaptation to an aquatic environment. Key words Brasenia schreberi; anatomy; histochemistry; morphology; primary

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structure; polysteles

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1. Introduction

The authors became interested in Brasenia schreberi J. F. Gmel. (Cabombaceae)

because it is a famous critically endangered aquatic plant species in China (Fu and Wiersema, 2001; Dong et al., 2010) and a basal angiosperm (APG, 2016). The shoots of B. schreberi have vertical and horizontal rhizomes which produce many

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adventitious roots, floating stolons and some leaves. Horizontal rhizomes and stolons often occur in dense rosettes on the vertical rhizomes and produce most leaves (Moseley et al., 1993; Williamson and Schneider, 1993; Fu and Wiersema, 2001); peduncles usually arise from stolons and support flowers and fruits (Richardson, 1969). The anatomy of B. schreberi in the Cabombaceae (Nymphaeales, basal angiosperms) has long been studied (e. g., Schrenk, 1888; Keller, 1893; Metcalfe and

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Chalk, 1950; Richardson, 1969). Stems were reported to have two bicollateral

vascular bundles embedded in aerenchyma (Schrenk, 1888; Zhang and Chen, 1988; Yu, 1996; Ding et al., 1997; Xu et al., 1999), and flower anatomy was reported in

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detail by Richardson (1969). Roots and rhizomes have been described as having

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helical or annular tracheary elements in primary xylem (Schneider and Carlquist, 1996; Carlquist and Schneider, 2009). Schneider and Carlquist (1996) first reported

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vessel elements in roots and rhizomes, but in 2009 Carlquist and Schneider reported that there are only tracheids in shoot tissues. While roots demonstrably have

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Casparian bands and suberin lamellae in endodermis and exodermis (Seago, 2002), in shoots, from Schrenk (1888) to Carlquist and Schneider (2009) there has never been

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adequate demonstration of the occurrence of barrier layer structures like endodermis with Casparian bands around vascular bundles and exodermis with Casparian bands

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and suberin lamellae.

Trichomes have been reported since the 1880s (Schrenk, 1888). Although glandular

trichomes were referred to as mucilage hairs by Carpenter (2006), like Wilkinson (1979), he considered them to be a type of hydropote because they had three cells, confirmed by scanning electron microscopy. Yet, Carpenter (2006, see page 668 - Fig 3, 4) compared and distinguished between mucilage hairs and hydropotes, which were 3

presumably absorptive of mineral salts and water (Wilkinson, 1979). Williamson and Schneider (1993, page 158), however, termed these trichomes “mucilage-secreting hairs”. During development, glandular hairs protrude from the epidermis and consist of stalk cells and an elongated glandular or hair cell, surrounded by mucilage and some cuticle (Schrenk, 1888; Keller, 1893; Zhou and Lu, 1985; Shi et al., 1991; Lu et al., 1995; Carpenter, 2006). Further, these trichomes have been reported not just on the epidermis (Carpenter, 2006), but also in the lacuna of aerenchyma (Schrenk, 1888;

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Ogden, 1974; Fahn, 1990) where they are clearly not epidermal in origin. We use the expression glandular trichomes for epidermal hairs that are covered in mucilage.

The important phylogenetic position of Brasenia among the angiosperms and basal

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angiosperms (APG, 2016; Soltis et al., 2018; Stevens, 2019), in particular, shows that a better understanding and knowledge of the anatomy of its shoots are needed to

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corroborate and build upon past findings. Further, with the prospects of the

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endangered B. schreberi losing its habitat in West Lake area, Zhejiang Province, and Lichuan County, Hubei Province, China (Dong et al., 2010), we wanted to provide a better anatomical basis of these shoot structures to provide a basis for understanding

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their possible intolerance of pollution in Lichuan county, China (Zhang et al., 2015). Accordingly, we report the anatomy of vertical rhizomes, horizontal rhizomes, stolons,

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peduncles, and leaves of B. schreberi.

2. Materials and Methods Brasenia schreberi samples were collected from cultivated aquatic plants in

Lichuan County, Hubei Province, China. All plants were fixed in FAA (Jensen, 1962) or were examined fresh. We sampled young and older segments of vertical rhizomes, horizontal rhizomes, floating stolons, peduncles, and leaves; young stem samples 4

were sectioned from visibly elongated internodes, and older vertical rhizome sections were taken from nodes with abscission layers, where stems and adventitious roots and leaves were sloughed off. All stem and leaf samples of B. schreberi were cut freehand with a two-edged razor blade under a stereoscope. Using anatomical and histochemical methods, sections were stained with Sudan red 7B (SR7B) for suberin lamellae (Brundrett et al., 1991), berberine hemisulfate–aniline blue (BAB) for Casparian bands and lignified walls

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(Brundrett et al., 1988; Seago et al., 1999), aniline blue for phloem (Peterson et al.,

2008), 2.5 µg/mL hoechst 33342 for nuclei (Pursel et al., 1985), and phloroglucinol– HCl (Pg) for lignin (Jensen, 1962). Specimens were examined under brightfield

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(Leica DME) and epifluorescence microscopy (IX71) and photographed with digital

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cameras (Nikon E5400).

Sample segments for ultrastructure of 3 mm × 3 mm × 1 mm were fixed for 3 h in

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2.5% glutaraldehyde, post-fixed in buffered 1% osmium tetroxide, and then dehydrated through an ethanol series. The samples were then embedded in Epon 812

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resin. Ultrathin sections were placed on copper grids and stained with uranyl acetate followed by lead citrate and examined in a JEX-1400 transmission electron

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microscope (JEOL; Wang et al., 2008; Guo and Liu, 2013).

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3. Results

Stems of Brasenia schreberi plants (Williamson & Schneider, 1993) consisted of

vertical rhizomes and horizontal rhizomes with adventitious roots, floating stolons arising from vertical or horizontal rhizomes, leaves arising from stem nodes, and peduncles arising from stolon nodes to produce flowers and fruits (Fig 1A, B, C). Where stolons became buried in the marsh substrate, they produced horizontal 5

rhizomes (Fig 1A, B). In older stages, horizontal rhizomes and stolons sloughed off from vertical rhizomes leaving distinct abscission scars or layers (Fig 1C). 3.1 Vertical rhizomes Using anatomical and histochemical methods, we have shown that short vertical rhizomes of B. schreberi had steles (Fig. 2A), which varied greatly. Most specimens had a partial ring of vascular tissue plus a large vascular bundle, with parenchymatous

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regions in between the vascular tissues (Fig 2A); we did not observe a complete ring of vascular tissue. Each vascular bundle or partial ring had external and internal

primary phloem around primary xylem. This arrangement could be interpreted as a

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polystele (as per Metcalfe and Chalk, 1950; Schmid, 1982) or as a solenostele (see Gifford and Foster, 1989). Bases of adventitious roots were usually evident in the

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short vertical axes (Fig 2A, 2C). Lignified primary xylem tracheids could be identified in longitudinal sections (Fig 2D) and in transverse sections between

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external and internal primary phloem (Fig 2B, 2C). Each bundle (Fig 2A) was surrounded by its own endodermis with Casparian bands (Fig 2B, 2E). Protoxylem

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lacuna were not easily identifiable in vertical rhizomes. Expansigenous aerenchyma, intercellular spaces or lacunae produced by cell division and expansion (Seago et al.,

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2005), occurred in a pith region, where lacunae were small, and in the cortex just peripheral to the vascular bundles (Fig 2A, B, C); the outer cortex had multiple layers

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of cells under the hypodermis (Fig 2A, 2G, 2H). Cells of the exodermal hypodermis or abscission layer (Fig 2A, F, G, H) had Casparian bands (Fig 2F), suberin lamellae (Fig 2G) and lignified cell walls (Fig 2H). Glandular hairs occurred on the epidermis (Fig 2A).

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3.2 Horizontal rhizomes, stolons and peduncles Unlike vertical rhizomes, horizontal rhizomes (Fig 3A, B) were distinctly polystelic with two bicollateral vascular bundles (Fig 3A, B), each with a central protoxylem lacuna (Fig 3A, B, C, Metcalfe and Chalk, 1950), between two groups of lignified primary xylem tracheids (Fig 3B, D, E) and interior to primary phloem (Fig 3B, D, F). Each vascular bundle of the polystele was surrounded by an endodermis with lignified Casparian bands (Fig 3D and inset, G, H). Protoxylem lacuna were usually distinctive

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between the two zones of visible tracheids (see Metcalfe and Chalk, 1950, page 67); the radial walls of the cells lining a lacuna fluoresced in a few specimens (Fig 3C), and fluorescent inner tangential walls lined other lacunae (Fig 3H); this tangential

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fluorescence might represent remnant tracheid cell walls stretched against cell walls during elongation. The sieve tube elements and companion cells of primary phloem

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had large numbers of small starch grains, whereas the cortex had larger grains in the

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inner cortex and usually fewer grains toward the center of the cortex (Fig 3F); sieve tube elements had sieve plates with numerous sieve pores (Fig 3I inset). The cortex had expansigenous aerenchyma (Fig 3A, B) with air spaces of various sizes encircling

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the vascular bundles of the polystele.

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Stolons (Fig 4A, B, C) and peduncles (Fig 4D, E, F) were also polystelic with two bicollateral vascular bundles and an endodermis with Casparian bands around each

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bundle (Fig 4C and inset, 4F and inset). Petioles (Fig 4G, H, I) had one bicollateral vascular bundle without an endodermis (Fig 4I). As in horizontal rhizomes, each bicollateral vascular bundle in stolons (Fig 4B, C), peduncles (Fig 4E, F) and petioles (Fig 4H, I) had a protoxylem lacuna between two regions of primary xylem near the lacuna and two regions of primary phloem, each distal to the lacuna. In petioles (Fig 4G), the singular bundle occurred in the centre of the axis. Dominant features of stems 7

and petioles were the various sizes of lacunae in the expansigenous aerenchyma (e.g., Fig 2A, 3A, 4A, D, G), including through the middle of the axis in stolons (Fig 4A) and peduncles (Fig. 4D). Peduncles (Fig. 4D) and petioles (Fig. 4G) had copious glandular trichomes. 3.3 Leaves and glandular trichomes The leaf blades had schizogenous palisade parenchyma and schizogenous and

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expensigenous spongy tissues (Fig 5A), and the abaxial epidermis had abundant glandular trichomes (Fig 5A, B); cuticle and palisade tissue were evident adaxially (Fig 5A, C). The vascular bundles had collateral bundles with a usually prominent

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protoxylem lacuna and primary xylem toward the adaxial side of a bundle (Fig 5A). Glandular trichomes were distributed on the surface of vertical rhizomes (Fig 2A),

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horizontal rhizomes (Fig 3A), stolons (Fig 4A), peduncles (Fig 4D), and leaves (petioles, Fig 4G, leaf blades, Fig 5A, B). Each glandular trichome had a storage

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space within a thin cuticle surrounding the glandular cell (Fig 5E); two lignified disklike stalk cells formed their base (Fig 5D, E inset, F), and we detected a nucleus in

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glandular cells (Fig 5D, G). The mucilage bubbles were quite large around the hairs (Fig 5B, D). The epidermis had a discontinuous cuticle; the cuticle (Fig 5H, I) was

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like a ring around the base of glandular trichomes (Fig 5H, I & insets), but it did not

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cover the protruding glandular cells at older stages.

4. Discussion In general, vertical rhizomes had varying vascular tissue arrangements, while

horizontal rhizomes, stolons, and peduncles of B. schreberi shoots had a primary structure with a distinct polystele of two vascular bundles characterizing the vascular system. Ground tissue like cortex and pith had primarily expansigenous aerenchyma, 8

and epidermis had glandular trichomes. Our findings for B. schreberi rhizomes and petioles were similar to the drawings by Ogden (1974), but differed for peduncles because Ogden (1974) illustrated four vascular bundles; we also did not find glandular trichomes within aerenchymatous lacuna that he noted, although he did not show such hairs in his drawings. Possibly there was a difference between B. scherberi plants in North America and China. Although vessel elements were earlier reported in roots and rhizomes of B.

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schreberi (Schneider and Carlquist, 1996), the vascular tissues of vertical rhizomes had only lignified tracheids, in agreement with Carlquist and Schneider (2009), although Richardson (1969, page 9) reported “few or no tracheary elements in

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the…stems”. In our specimens, vertical rhizomes had three or four vascular bundles often organized like a solenostele, and horizontal rhizomes, stolons, and peduncles

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had two bicollateral vascular bundles organized as polysteles; each vascular bundle

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was surrounded by an endodermis characterized by lignified Casparian bands. In standard microscopic methods, Richardson (1969, pages 9, 51) reported and

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illustrated an endodermis with Casparian bands in Brasenia stems, but did not find an endodermis in peduncles or petioles. We agree that petioles had one bicollateral

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vascular bundle without an endodermis. In having only Casparian bands, the endodermis surrounding polysteles of B. schreberi stems differed from its roots which

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have additional suberin lamellae walls in its cells (Seago, 2002). These vascular and endodermal features in Brasenia may be very important for basal angiosperms and Cabombaceae, but Moseley et al. (1984) did not mention shoot endodermis or show images of it in the closely related Cabomba. Stems, peduncles, and even petioles may have endodermis with Casparian bands in other aquatic or wetland plants like Typha,

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Artemisia, Ranunculus trichophyllus and Hydrocotyle sibthorpioides (McManus et al., 2002; Vecchia et al., 1999; Yang et al., 2015; Zhang et al., 2017, 2018). The characteristic protoxylem lacuna, derived from protoxylem degeneration according to Metcalfe and Chalk (1950), sometimes displayed a unique fluorescence in radial walls of the surrounding cells, but we do not interpret these to be Casparian bands and endodermis, as claimed for Isoetes leaves by Troiád et al. (2000); these are parenchyma cells adjacent to apparently lysed protoxylem elements and may

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contribute to maintenance of the protoxylem lacuna. Richardson (1969, pages 8-9) did not consider these to be protoxylem lacuna, but instead to be a “schizolysigenous

lacuna”…. surrounded by a uniform layer of cells (ring-like in transection) which

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possibly serves as a passageway for water and minerals”. However, protoxylem

lacuna are generally just spaces where remnant cell walls can only occasionally be

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found. The cortex had honeycomb-shaped lacunae or expansigenous aerenchyma in B.

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schreberi shoots like its roots and roots in other Nymphaeales (Seago, 2002) and Acorales (Seago et al., 2005) and in some shoots of other species (Jung et al. 2008).

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Schrenk (1888) and Keller (1893) described that glandular trichomes had a storage space or sac around the main glandular cell and had one or two stalk cells (Shi et al.,

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1991; Yu, 1996; Zhou and Lu, 1985). We observed that each glandular trichome in B. schreberi had one large cell with storage space and a thin cuticle except at older

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stages, when they were often absent, and the base had two lignified and lipoidal disklike stalk cells (see Carpenter, 2006). It should be noted that storage space surfaces of glandular trichomes were covered with cuticle and subcuticle or cell walls in tomato, peppermint, Cannabis sativa, Artemisia annua, Humulus lupulus, Cordia verbenacea, (Mahlberg and Kim, 1991; Duke and Paul, 1993; Kim and Mahlberg, 2000; Turner et al., 2000; Ventrella and Marinho, 2008; Tissier et al., 2017). The thin cuticle of 10

storage spaces and discontinuous cuticle of epidermis may contribute to B. schreberi sensitivity to water pollution (Zhang et al., 2015). In our specimens, we did not find the very small structures termed hydropotes (see Wilkinson, 1979) which Carpenter (2006) reported with illustrations in B. schreberi; instead, we found that the abaxial leaf surface and other shoot surfaces were densely covered with the mucilaginous glandular trichomes or their remnants that gave the plant the very mucilaginous appearance and character. In her review of structures on

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the leaf surface, Wilkinson (1979) had noted that the trichome-like structures

produced by epidermal cells were hydropotes and were at first “mucilage-secreting hairs”, but later assume “the function of absorbing water and mineral salts”

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(Wilkinson, 1979, p 163), although the evidence for that is scant. Williamson and

Schneider (1993, p 158) did report “occasional hydropote-like cells and numerous

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mucilage-secreting hairs” or trichomes. Unlike Fahn (1990) and Ogden (1974) who

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reported mucilaginous cells in aerenchymatous lacuna in B. schreberi plants from New York, USA, without illustration, we did not observe such hairs or glandular

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trichomes in aerenchymatous lacuna.

The abscission layers, or exodermal hypodermis, on vertical rhizomes had

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Casparian bands, suberin lamellae and lignin. Genlisea also had a discontinuous cuticle for glandular hairs (Plachno et al., 2005). The cuticles were continuous in the

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stems of wetland plants like Typha (McManus et al., 2002), R. trichophyllus and H. sibthorpioides (Vecchia et al., 1999; Yang et al., 2011, 2014, 2015). Lignified and suberized peripheral mechanical rings and periderms occurred in Cynodon dactylon, Paspalum distichum, Artemisia and Zizania latifolia stems (Yang et al., 2011, 2014; Zhang et al., 2017, 2018).

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There are developmental and structural issues beyond the scope of this study that could use future research, for example, detailed protoxylem ontogeny and possible function of its surrounding cells, nature of the complex vasculature in the vertical rhizome, nodal structures and the structure of the accompanying endodermis, and the transformation of the exodermal hypodermis into an abscission layer that functions when branches and petioles abscise. In conclusion, shoot morphology of B. schreberi displayed vertical and horizontal

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rhizomes, stolons, peduncle, and leaves with petioles and blades; anatomically, stems had a polystelic vascular configuration and endodermis with Casparian bands around

each vascular bundle, but leaves did not have an endodermis around vascular bundles.

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The cuticle was discontinuous on the epidermis, glandular trichomes had one large glandular cell with spaces, and the base had two disk-like stalk cells with a thin

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cuticle; mucilage characterized plant surfaces. Air spaces included protoxylem lacuna

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and mostly expansigenous aerenchyma in the ground tissues. These structural features of B. schreberi were consistent with basal angiosperm traits and their adaptation to

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aquatic environments.

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Conficts Of Interest

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There is no any conficts of interest or disclose.

Acknowledgments

This work was supported by funding from the Engineering Research Center of Ecology and Agriculture Use of Wetland, Ministry of Education opening fund, 12

Yangtze University (KF201603), and Key Laboratory of Aquatic Botany and Watershed Ecology opening fund (2017), Wuhan Botanical Garden, The Chinese

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Academy of Sciences.

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Legends:

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Fig. 1. A-C, Photographs of Brasenia schreberi vegetative organs. scale bar = 5 cm.

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1A. aged plant, vertical rhizomes, horizontal rhizomes, stolons, leaves, in vivo; 1B. stolons, peduncle, leaves, fruits, in vivo; 1C. aged vertical rhizomes, abscission layers (arrowheads), stolons, adventitious root, in vivo. Abbreviations in all figures: ae, aerenchyma; al, abscission layer; ar, adventitious roots; BAB, berberine hemisulfate–aniline blue stained; bvb, bicollateral vascular 20

bundles; cc, companion cells; co, cortex; cu, cuticle; ep, epidermis; fr, fruits; gc, glandular trichome; gu glandular cell; hr, horizontal rhizomes; la, protoxylem lacuna; le, leaves; pe, peduncle; ph, primary phloem; Pg, phloroglucinol–HCl stained; ps, polystele; pt, palisade tissue; sc, stalk cell; se, sieve elements; sp, sieve plates; SR7B, Sudan red 7B stained; st, stolons; TBO, toluidine blue O stained; TEM, transmission

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electron microscopy; vr, vertical rhizomes; vb, vascular bundles; xy, primary xylem.

Fig. 2. A-H, Photomicrographs of Brasenia schreberi old vertical rhizomes. scale bar

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=50 um.

2A. Transverse section with stolon sloughed off at abscission layer, polystele,

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aerenchyma, cortex, adventitious root bases, TBO; inset, glandular hairs from the epidermis, SR7B;

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2B. Transverse section with one vascular bundle of polystele, aerenchyma, endodermis (arrowheads), primary xylem, primary phloem, TBO;

2C. Transverse section with adventitious root bases (arrow) connected to polystele, aerenchyma, TBO; 2D. Longitudinal section with tracheids (arrowhead), BAB;

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2E. Transverse section with a portion of a vascular bundle, aerenchyma, Casparian bands in the endodermis (arrowhead), BAB; 2F. Transverse section with Casparian bands (arrowhead) in cell walls of abscission layer, BAB; 2G. Transverse section with suberin in cell walls of abscission layer (arrowhead), cortex, SR7B; 2H. Transverse section with lignin in cell walls of abscission layer (arrowhead),

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cortex, Pg.

Fig. 3. A-I. Photomicrographs of old horizontal rhizomes and stolon in Brasenia schreberi. scale bar =50 um.

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3A.Transverse section of horizontal rhizome, bicollateral vascular bundles with protoxylem lacuna (space in vascular bundle), aerenchyma, cortex, inset shows glandular hairs on the epidermis, TBO, in vivo; 3B. Transverse section of horizontal rhizome, a part of vascular bundle; endodermis (arrowhead), protoxylem lacuna, primary xylem, primary phloem (*), aerenchyma, TBO; 3C. Transverse section aged horizontal rhizome, protoxylem lacuna, fluorescing radial

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cell walls in cells around lacuna (arrow), BAB; 3D. Transverse section horizontal rhizome, Casparian bands in the endodermis

(arrowheads), primary phloem (*), primary xylem, inset shows enhanced Casparian

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bands (arrow), BAB;

3E. Longitudinal section horizontal rhizome, tracheids (arrowhead), BAB;

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3F. Longitudinal section horizontal rhizome, grains in primary phloem (arrowhead),

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grains in cortex (arrow), aniline blue stained viewed under brightfield; 3G. Transverse section horizontal rhizome, lignified endodermis (arrowhead), vascular bundle, Pg;

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3H. Transverse section stolon, protoxylem lacuna with remnant tracheid cell wall (arrow), Casparian bands in the endodermis (arrowhead), BAB;

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3I. Transverse section horizontal rhizome, sieve elements, sieve plates, companion

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cells, aniline blue stained, inset shows sieve plate under brightfield.

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Fig.4. A-I, Photomicrographs of Brasenia schreberi shoots. scale bar =50 um.

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4A. Transverse section stolons, bicollateral vascular bundles, aerenchyma, cortex, TBO, inset shows glandular hairs on the epidermis, SR7B, in vivo; 4B. Transverse section bicollateral vascular bundle of stolons, protoxylem lacuna,

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primary xylem, primary phloem (*), aerenchyma, TBO, in vivo; 4C. Transverse section stolon, protoxylem lacuna, Casparian bands on the

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endodermis (arrowhead), primary xylem, primary phloem (*), aerenchyma, inset is

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enhanced to brighten Casparian bands (arrow), BAB; 4D. Transverse section peduncle, bicollateral vascular bundles, aerenchyma, cortex, glandular trichomes, unstained, in vivo;

4E. Transverse section bicollateral vascular bundle of aged peduncle, protoxylem lacuna, primary xylem, primary phloem (*), aerenchyma, TBO;

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4F. Transverse section of peduncle, protoxylem lacuna, Casparian bands on the endodermis (arrowheads), primary xylem, aerenchyma, inset shows enhanced Casparian bands (arrows), BAB, in vivo; 4G.Transverse section of petiole, bicollateral vascular bundle, aerenchyma, cortex, glandular trichomes, TBO; 4H. Transverse section bicollateral vascular bundle of petiole, protoxylem lacuna, primary xylem, primary phloem (*), aerenchyma, TBO;

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(*), primary xylem, aerenchyma, BAB, in vivo.

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4I. Transverse section of petiole vascular bundle, protoxylem lacuna, primary phloem

Fig. 5. A-I. Photomicrographs of leaf blade, glandular trichomes of shoots in Brasenia schreberi. scale bar =50 um, except where note. 25

5A. Transverse section leaf blade, vascular bundle with protoxylem lacuna, remnants of glandular trichomes, palisade tissue; aerenchymatous spongy tissue; epidermis, SR7B, in vivo; 5B. Transverse section abaxial leaf blade surface, glandular trichomes (arrowhead), mucilage (*), SR7B, in vivo; 5C.Transverse section adaxial leaf blade, palisade tissue, cuticle, BAB; 5D. Lateral view of glandular trichomes, glandular cells (arrowhead), cuticle connects

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to the stalk cell (arrow), mucilage (*), BAB, in vivo; 5E. Cuticle apart from the edge between stalk cell and glandular cell to form storage space (*), lignin in walls (arrowhead), TEM; inset shows lignified cell walls

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(black arrowhead), Pg;

5F. Glandular trichome composed of glandular cell and stalk cells, epidermis, cuticle

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(arrowhead), TEM;

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5G. Single nucleus of glandular cell (upper arrowhead), stalk cell, single nucleus of epidermis (lower arrowhead), hoechst 33342 stained, scale bar = 25 μm, in vivo; 5H. Transverse section glandular trichomes, epidermis, cuticle, SR7B;

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5I. Transverse section glandular trichomes, the ring-like, and dot-like cuticle of the glandular trichome bases, two upper insets are young stage, two lower insets are

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older stages, scale bar = 25 μm, BAB.

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