Structure and ion physiology of Brasenia schreberi glandular trichomes in vivo

Brasenia schreberi is a critically endangered aquatic basal angiosperm. In this work, we characterized the structure of the glandular trichomes of B. schreberi morphologically and histochemically. We used a variety of structural, histochemical and permeability stains for the characterization, and we tested the effects of stress in vivo using NaCl and ethanol. We observed that the glandular trichome of B. schreberi are composed of two disk-like stalk cells, and a glandular cell which surround a cuticular storage space. The cuticle is discontinuous at the surface of the shoots. Nearly half of young trichomes senesced in 0.9% NaCl, and mature trichomes senesced at 1.8% NaCl. About half of young trichomes senesced under 3% ethanol and mature trichomes senesced in 2% ethanol after 20 min of treatment. The physiology of glandular trichomes affects the way they secrete mucilage via storage space at a young stage. The trichomes become permeable and absorb ions when mature. This transition depends on the osmiophilic material and the dynamic protoplast. It can accelerate senescence and disassembly by ion accumulation. Permeability tests and ion treatments of glandular trichomes provide new insights for fertilizer research. Our study highlights the structure and physiology of B. schreberi glandular trichomes.


INTRODUCTION
The water-shield, Brasenia schreberi J. F. Gmel. (Cabombaceae, Nymphaeales), is a well-known basal angiosperm plant (Williamson & Schneider, 1993;The Angiosperm Phylogeny Group, 2016). In China, it is listed as endangered as a result of habitat loss (Fu & Wiersema, 2001;Zhang et al., 2015). It is eaten as a vegetable and used as a medicinal plant. Its compounds have anti-inflammatory effects and can inhibit HIV-1 reverse transcriptase and viral replication (Legault et al., 2011;Hisayoshi et al., 2015). The gross morphology of the plant consists of vertical and horizontal rhizomes, floating stolons with peduncles, and adventitious roots (Moseley et al., 1984;Williamson & Schneider, 1993). The shoots are covered on the surface with hydrophilic lubrication mucilage (Wilkinson, 1979;Liu et al., 2014). Some of its anatomical features have been thoroughly studied, including the adventitious roots (Seago, 2002;Seago et al., 2005), stems with leaves (Xu et al., 1999),
We used 2.5 mg/mL Hoechst 33342 to stain nuclei for 10 min with 350 nm excitation and detected it at 455-465 nm (Pursel et al., 1985). These sections were observed and photographed using a Leica-SP8 confocal microscopy (Leica Camera, Wetzlar, Germany).

Ultrastructure of transmission electron microscopy
Sections of 2 Â 2 Â 1 mm were fixed for 3 h in 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 resin. Ultrathin sections were placed on copper grids and stained with uranyl acetate followed by lead citrate and examined with a JEX-1400 transmission electron microscopy (JEOL, Tokyo, Japan) (Guo & Liu, 2013;Wang et al., 2008).

NaCl and ethanol treatments
Four concentrations of NaCl solutions (0%, 0.9%, 1.8%, 2.7% w/v) and five concentrations of ethanol (0%, 1%, 2%, 3%, 4% v/v) were used to stress glandular trichomes at young and mature stages with six replicates per treatment. Sections of fresh petioles with mucilage were immersed in the above NaCl and ethanol solutions for specific amounts of time: 0, 10, 20, and 30 min. There were 216 experimental sections. After treatment, plant sections were rinsed five times with deionized water, lightly blotted, and sections were mounted in deionized water to be viewed under blue light and then photographed. The numbers of senescent (or aged) trichomes with ball-like, pillar-like, foam-like, or deformed protoplasts were counted as aged.

Statistical analyses
We quantified the number of senescent trichomes that were stressed by the different concentrations and timing of the NaCl and ethanol treatments in the young and mature samples. The senescence of glandular trichomes is expressed as percentages under various NaCl and ethanol concentrations for treatment of different durations in young and mature stages were first transformed based on square root arcsine to normalize the data and then subjected to ANOVA using SAS 9.1 software (SAS Institute, Cary, NC, USA) and the means with standard deviations were compared using Duncan's multiple range test at the <5% level. Graphs were plotted using original non-transformed values. Data are means of 36 samples.
The elongated glandular cell is one cell and it is surrounded by storage space (Figs. 1D-1F). The surface of the storage space has a thin cuticle that contains osmiophilic materials in its inner face with affinity for Hoechst 33342 (Figs. 1G-1I). The walls of the glandular cell have thin cuticle and osmiophilic materials, and affinity to Hoechst 33342 (Figs. 1A, 1D, 1F and 1J) in the mature stage after storage space sloughed off. At the base of glandular trichomes, stalk cells have a cuticle and lignin (Figs. 1C and 1K). The epidermis has a discontinuous cuticle that is like a ring around the base of glandular trichomes (Fig. 1K),

Dynamic protoplast of glandular trichomes
The protoplast of the glandular cells appeared smooth, spotted, and had short nodeand pillar-like structures at young and mature stages when viewed under brightfield, epifluorescence and autofluorescence (Figs. 2B, 2D, 3A and 3B), or partly turned brown in color (Fig. 2E). The aged glandular cells turn black or have pillar-like, ball-like and twisted protoplast, and slough off with small new glandular cells, and some mucilage on stems and petioles (Figs. 2C, 2E-2H and 3C-3E). Effect of NaCl stress on glandular trichomes Protoplasts appeared smooth in young glandular cells that were stressed by the 0.9% NaCl solution for 10 min (Fig. 4A). Almost half (45.8%) of the young glandular cells senesced after 20 min of treatment with 0.9% NaCl (Fig. 5A) and pillar-like structures were observed in aged glandular cells. Moreover, ca. 82.1% of all young glandular cells had pillar-like structures after 30 min of treatment with 0.9% NaCl (Figs. 4B and 5A). Mature glandular cells had spotted protoplasts after being stressed by 1.8% NaCl for 10 min (Fig. 4C). Almost half (47.9%) of senesced protoplasts formed a foam-like structure after 20 min of treatment of mature glandular cells with 1.8% NaCl (Fig. 5B), and approximately 89.5% of mature glandular cells had a foam-like protoplast after 30 min of treatment with 1.8% NaCl, which was significantly more than at 20 min post treatment with the same concentration of NaCl (Figs. 4D and 5B). More than half (54.0%) of mature senesced glandular cells had foam-structures and shrank or had heavily twisted protoplasts after being stressed by 2.7% NaCl for 10 min  5B). We observed a disassembly of the protoplast in glandular cells when stressed by 2.7% NaCl for 20 min (Fig. 4E).

Effect of ethanol stress on glandular trichomes
Across all ethanol treatments, we observed glandular cells senesced and disassembled compared with the control. The 1% ethanol treatment resulted in senescence of fewer glandular cells than in the untreated control group in the short term (0-10 min) among young glandular cells (Figs. 6A, 6B and 7A). When we looked at the effect of ethanol

Structure of glandular trichomes
In our study of the glandular trichomes of B. schreberi, we observed that these trichomes consist of two disk-like stalk cells and a glandular cell surrounding a storage space covered with a thin cuticle at the epidermal surface of stems, petioles, and leaves. Schrenk (1888) and Keller (1893) reported that glandular trichomes emerged from the epidermis and had two disk-like stalk cells and a glandular cell with one or two heads and surrounded a sac or storage space, and the glandular cell had three layers of cuticle (Zhou & Lu, 1985;Shi et al., 1991). In our study, we observed the glandular cell had a single layer of cuticle. The cuticle and subcuticle or cell walls cover the storage space surface of glandular trichomes, such as in Artemisia annua (Duke & Paul, 1993), Cajaninae (De Vargas et al., 2019), Calceolaria volckmanni (Sacchetti et al., 1996), Cannabis sativa (Mahlberg & Kim, 1991), Cordia verbenacea (Ventrella & Marinho, 2008), Doronicum  structures in the young and mature stages. In the aged stage, the protoplasts have pillar-or ball-like, structures and are twisted; they then turn black and slough off (Schrenk, 1888).
In our specimens, we did not find the very small structures termed hydropotes (Wilkinson, 1979) which Carpenter (2006 reported with illustrations in B. schreberi; instead, we find that the abaxial leaf surface and other shoot surfaces are densely covered with glandular trichomes that give the plant a very mucilaginous appearance. Wilkinson noted that the trichome-like structures produced by epidermal cells were hydropotes and were at first "mucilage-secreting hairs," but later assumed the function of "absorbing water and mineral salts" (Wilkinson, 1979;Schrenk, 1888). While Carpenter (2006) explicitly stated that trichomes were lacking in Brasenia, Williamson & Schneider (1993) did report "occasional hydropote-like cells and numerous mucilage-secreting hairs" or trichomes. Ogden (1974) and Fahn (1990) reported such hairs in lacunae from New York, US, B. schreberi, but we did not observe glandular trichomes in aerenchymatous lacunae.
The glandular trichomes we observed in B. schreberi represent a simple type consisting of two stalk cells at base and a glandular cell head in plant trichomes (Carpenter, 2006;Huchelmann, Boutry & Hachez, 2017;Tissier, Morgan & Dudareva, 2017). Multicellular trichomes have one or more cells secreting trichome head, stalk cells, intermediate cells, and basal cells, such as peppermint, sunflower and tomato (Turner, Gershenzon & Croteau, 2000;Bergau et al., 2015;Amrehn et al., 2016). A cuticle surrounds the base of the B. schreberi glandular trichomes, and is discontinuous with the epidermal surface in aged samples. This epidermal cuticle is similar to that observed in the genus Genlisea (Lentibulariaceae), which has a discontinuous cuticle (Plachno, Faber & Jankun, 2005). The discontinuous cuticle is unlike the tight barriers that are in the peripheral mechanical ring, periderm and cuticle-structures that are common in wetland plants along the Yangtze River, such as Alternanthera philoxeroides (Yang et al., 2019), Artemisia lavandulaefolia (Zhang, Yang & Seago, 2018), Cynodon dactylon (Yang et al., 2011), Phalaris arundinacea (Zhang et al., 2017a), and Zizania latifolia .

Ion permeability of glandular trichomes
We used berberine hemisulfate as a permeability trace to assess the ionic permeability of the apoplastic barrier layers of the glandular trichomes. Barriers to absorption typically block berberine from entering inner organs for tests with Iris (Meyer, Seago & Peterson, 2009), Artemisia lavandulaefolia (Zhang, Yang & Seago, 2018), and Alternanthera philoxeroides (Yang et al., 2019). The glandular trichomes of B. schreberi secrete mucilage via storage space when young, then transfer that mature stage to be able to absorb greater numbers of ions. Ion permeability ceases at the oldest stages, and in our study Hoechst 33342 staining showed that ions can accumulate both in the cuticle of the storage space and glandular cell. Glandular trichomes are known to secrete mucilage, but this study is the first to observe that trichomes can absorb ions.
The young glandular cells were more sensitive than mature glandular cells to the NaCl treatments. Nearly half of young glandular cells senesced in the 0.9% treatment, whereas an equal number of mature cells senesced in the 1.8% treatment. Aged glandular cells that senesced showed pillar-like, foam-like and obscure structures. In our ionic permeability test, mature glandular cells absorbed more ions, which may explain why mature glandular cells are more tolerant than young glandular cells to NaCl stress.
Young glandular cells were more tolerant than mature glandular cells to stress with ethanol solutions. Nearly half of young glandular cells senesced under the 3% treatment, whereas nearly half of mature glandular cells had senesced in the 2% treatment at the same time point. Aged glandular cells also showed pillar-like, foam-like, and obscure structures. NaCl and ethanol stress showed that protoplasts have a dynamic, complex organelle membrane system and that young glandular cells are more tolerant to ethanol stress than mature glandular cells. The protoplasts of both young and mature glandular cells appeared to have similar pillar-like, foam-like, and obscure structures in the NaCl and ethanol treatments.
Inorganic and organic ions penetrated the storage space and glandular cell and intensely absorbed the Hoechst 33342 stain in trichomes that were at the mature stage in both the berberine permeability test, and the NaCl and ethanol stress tests. The trichomes cuticles contain osmiophilic material in Artemisia annua, Cannabis sativa, and Doronicum (Mahlberg & Kim, 1991;Duke & Paul, 1993;Muravnik, Kostina & Mosina, 2019). Glandular trichomes are permeable and absorb ions that may contribute to the osmiophilic material in the cuticle of trichomes and dynamic protoplasts. High concentrations of NaCl and ethanol solutions accelerated the senescence of glandular trichomes following just short periods of exposure, which provide new insight that may facilitate fertilizer research (Fu & Wiersema, 2001;Zhang et al., 2015). We suggest that lower concentrations of fertilizer should be used in the water of B. schreberi fields.

CONCLUSIONS
Our study revealed that the glandular trichome of B. schreberi is composed of stalk cells and a glandular cell surrounding a storage space which is covered with a thin cuticle on the epidermis. The cuticle is discontinuous at the shoots surface. The physiological function of glandular trichomes secrete mucilage via storage space at young stage, then become permeable and absorb ions at the mature stage which depends on the osmiophilic material and the dynamic protoplast, and accelerate senescence and disassembly by ion accumulation. Our analysis of the morphological and physiological characteristics of B. schreberi glandular trichomes may facilitate understanding of this endangered plant's taxonomy, evolution and phylogeny. Permeability tests and ion treatments of glandular trichomes may provide new insight for future fertilizer research (Fu & Wiersema, 2001;Zhang et al., 2015).