Revisiting hydropotes of Nymphaeaceae: ultrastructural features associated with glandular functions

Hydropotes are specialized epidermal structures involved in water and mineral flux into and out of the plant body. We analyzed hydropote morphology of four species of Nymphaeaceae: Nymphaea caerulea, Nymphaea lotus , Nymphaea rubra , and Victoria amazonica . Leaf samples were processed following conventional techniques for plant anatomy and for scanning and transmission electron microscopy. We observed hydropotes comprising an elongated apical sharp-pointed portion with a base composed of two to three short specialized cells. In a later developmental stage the apical sharp-pointed portion was detached and the mature hydropotes comprised an upper lens-shaped cell, a bowl-shaped cell and a large foot cell. Ultrastructural analysis revealed that the lens-shaped cell possesses labyrinthine projections in its outer periclinal wall and abundant plasmodesmata in its inner periclinal wall. Several mitochondria were present in the cytoplasm of both the lens-shaped and the bowl-shaped cells. The cytoplasm of the foot cell was reduced and possessed plastids with starch grains. The ultrastructure of the hydropotes is typical of cells involved in transporting substances and corroborates their role in the flux of substances into and out of the cell. These findings contribute to a better understanding of the functioning of hydropotes and shed light on this little-explored issue.


d and the bow
-shaped cells.The cytoplasm of the foot cell was reduced and possessed plastids with starch grains.The ultrastructure of the hydropotes is typical of cells involved in transporting substances and corroborates their role in the flux of substances into and out of the cell.These findings contribute to a better understanding of the functioning of hydropotes and shed light on this little-explored issue.

Introduction

Plants communicate with the environment through specialized epidermal structures, which are involved in the maintenance of their essential physiological and biochemical processes (Carpenter 2006) and in their establishment in the most diverse environments (Dickson 2000;Javelle et al. 2011).Hydropotes (water drinkers) are epidermal appendices specialized for life in an aquatic environment (Fahn 1979).They are present on the abaxial surface of floating leaves (Fahn 1979;Wilkinson 1979;Catian & Scremin-Dias 2013) and reproductive floating organs (Zini et al. 2017;Coiro & Lumaga 2018) of some species of aquatic plants such as those belonging to Aponogetonaceae, Menyanthaceae, Potamogetonaceae, Nymphaeaceae, Alismataceae (Lavid et al. 2001), and Cabombaceae (Borsch et al. 2008).

These glandular epidermal structures are involved in water and mineral transport into and out of aquatic plants (Fahn 1979;Mishra & Dubey 2010), and are able to retain more ions than common epidermal cells (Lüttge et al. 1971).Due to these functional characteristics, plants bearing hydropotes have shown great potential for phytoremediation (L vid et al. 2001), as is the case with species of Nymphaea.It has been shown that when exposed to cadmium, for example, hydropotes are able to trap cadmium-crystals through the activities of peroxidase and polyphenol oxidase (Lavid et al. 2001).However, despite their ecological importance for plants growing in aquatic environments, the morphological and subcellular features of hydropotes remain poorly-known.

The presence of hydropotes is widespread in Nymphaeales (Carpenter 2006).Lüttge & Krapf (1969) described the hydropotes of species of Nymphaeaceae as comprising four cells: a cap cell, a lens-shaped cell, a cup-like cell and a basal cell.These authors also observed small vacuoles filled by dark electron-dense material and walls forming protuberances in the lens-shaped cell and cup-like cell.Later, Carpenter (2006) described hydropotes with a hair-like portion and, in discussing the distribution and homology of epidermal structures among basal angiosperms, showed the presence of hydropotes in Nuphar, Nymphaea, Euryale, Victoria (Nymphaeaceae) and Brasenia (Cabombaceae).In the same study, Carpenter (2006) described hydropotes as comprising three cells: a lens-shaped cell, a bowl-shaped cell and a foot cell (basal).For the plants naturally occurring in aquatic environments in Brazil, the literature reports the resence of hydropotes only in the floating organs of Nymphaea species (Catian & Scremin-Dias 2013;2015).For these plants, Catian & Scremin-Dias (2013) reported hydropotes con

medial cell, an
an apical cell deciduous part.Nevertheless, detailed morphological studies focusing on the hydropotes of Brazilian aquatic plants, mainly involving ultrastructural analysis, are lacking.

In this study we characterized in detail the structure and the ultrastructure of hydropotes found in species of Nymphaeaceae in order to better understand their functioning.


Materials and methods


Plant material

We studied Nymphaea lotus L., N. rubra Roxb.ex Salisb., N. caerulea Savigny and Victoria amazonica (Poepp.)J.C. Sowerby.Samples of fully expanded leaves were collected from adult plants (n = 3) living in an artificial lake in the Ba

), dehydrated in an ethanol series, and
embedded in Leica® historesin.Cross sections (5-μm thick) were stained with 0.05 % toluidine blue, pH 4.3 (O'Brien et al. 1964), and mounted in Entellan® on permanent slides.The material was analyzed and the relevant results documented using a photomicroscope Leica® (model DMR, Germany) with a digital camera Leica® (model DFC 425).


Scanning electron microscopy (SEM)

Samples of leaves were fixed in 2.5 % glutaraldehyde with 0.1 M sodium phosphate buffer, pH 7.3, overnight at 4 °C.The samples where then dehydrated in acetone series, cr

ical-poi
t dried using a CPD 030 -Leica (Germany), mounted on aluminum stubs, gold-coated (Robards 1978), and examined with a Quanta 200 -FEI Company (USA) scanning electron microscope.


Transmission electron microscopy (TEM)

For transmission electron microscopy, samples of leaves of N. lotus and V. amazonica were fixed in 2.5 % Karnovsky in 0.1 M sodium phosphate buffer, pH 7.3, for 24 h; postfixed with 1 % osmium tetroxide in the same buffer for 1 h; dehydrated in an acetone series; and embedded in Araldite® resin.Ultra-thin sections were obtained with ultramicrotome EM UC7 -Leica (Germany) and stained with uranyl acetate and lead citrate (Reynolds 1963).The samples were examined using a Tecnai Spirit -FEI Company (USA) transmission electron microscope.


Results

In all studied species, hydropotes in two different developmental stages were observed side by side in the expanded leaves (Fig. 1A-B).The young hydropotes were morpho ogically similar to non-glandular trichomes, being comprised of an elongated sharp-pointed apical portion with 1-4 cells and base composed of two short cells (Fig. 1C).In a later developmental stage the short cell just below the sharp-pointed apical portion divided giving rise to an upper lens-shaped cell (Fig. 2A-B) and a subjacent bowl-shaped cell; the sharp-pointed apical portion then started to senesce and detached from the lens-shaped cell (Fig. 2C-D).Debris of the detached sharp-pointed apical portion were observed on the lens-shaped cell in lesser (Fig. 2D) or greater (Fig. 2E) amounts.Mature hydropotes (Fig. 2E) were short and contained an upper lens-shaped cell, a bowl-shaped cell and a large foot cell (Fig. 2F-I).Although the general structure of the hydropotes was similar among the studied species, the shape and position of the cells varied.In V. amazonica the foot cell was oval, and the bowl-shaped cell was very narrow and inserted in a level below the common epidermal cells (Fig. 2F).In the three Nymphaea species studied the foot cell was rectangular to square, and the bowl-shaped cell was located above the common epidermal cell level (Fig. 2G-I).

The ultrastructural studies reveal that hydropotes of both studied species, N. lotus and V. amazonica (Figs. 3, 4), were similar.Debris of the sharp-pointed apical portion could be seen above the lens-shaped cell (Fig. 3A, B).The lens-shaped cell possessed thick anticlinal and outer periclinal pectin-cellulosic walls.Labyrinthine projections were observed in the outer periclinal wall (Fig. 3C, D); in such cell wall projections, the cellulose microfibrils showed loose arrangement (Fig. 3E).Numerous plasmodesmata occurred in the inner periclinal wall (Fig. 3C, Fig. 4A) connecting the lens-shaped cells to the bowl-shaped cells.The plasma membrane had an irregular contour; and the cytoplasm was dense and abundant (Fig. 3C-D) with several mitochondria with dilated cristae (Fig. 3D).Abundant free ribosomes and   polyribosomes were present in the cytoplasm (Fig. 3F).The bowl-shaped cell was thin-walled; labyrinthine projections were seen in the inner pericli

l wall of o
ly N. lotus (Fig. 3C).The cytoplasm was abundant and clearer (Fig. 4A-F) than in the lens-shaped cell; the nucleus was elongated with irregular contour (Fig. 4B).In the cytoplasm, the mitochondria were abundant, voluminous with dilated cristae (Fig. 4C-E); electron-dense inclusions were observed in the mitochondria (Fig. 4E).It was remarkable the proximity and apparent connection between mitochondria (Fig. 4C-E, F) in these cells.Polyribosomes and free ribosomes occurred in the cytoplasm (Fig. 4B, E, F).Short profiles of rough and smooth endoplasmic reticulum (Fig. 4F) were visualized in these cells.Small vacuoles with membranous and flocculent inclusions we e observed in the cytoplasm of these cells (Fig. 4A).The foot cell possessed thin pectin-cellulosic walls, reduced cytoplasm and developed vacuome (Fig. 4A) The plasma membrane had an irregular contour.The cytoplasm was clear and possessed mitochondria with dilated cristae, plastids with starch grains with signs of degradation (Fig. 4C-D) and plastoglobuli (Fig. 4C); free oil droplets were present in the cytoplasm (Fig. 4B); vesicles were observed fusing to the plasma membrane (Fig. 4D).


Discussion

Our structural and ultrastructural results corroborate the information available in the traditional literature on the hydropotes in Nympheaceae (Lüttge 1964;Lüttge & Krapf 1969;Fahn 1979) and add novelties to this topic.Here, we did not disregard the sharp-pointed apical portion of young hydropotes and we proposed differential functions to hydropotes according to their distinct shape in different developmental stages.The origin of the lens-shaped and the bowl-shaped cells is here demonstrated by the first time.We were able to suggest different routes of substance transport along the hydropote body according to the ultrastructural features of their cells.

We observed hydropotes both with and without a sharppointed apical portion situated side-by-side in expanded leaves of N. lotus, N. rubra, N. caerulea and V. amazonica.Hydropotes with and without a sharp-pointed a ical portion seem to represent different developmental stages of the same glandular structure.In all of these species, we observed the sharp-pointed apical portion of some hydropotes to be senesced and detached, with just the basal portion intact.We considered this basal portion to be a mature hydr pote, which represents the most common hydropote structure described in literature (Lüttge 1964;Lüttge & Krapf 1969;Fahn 1979).Hydropotes with and without a sharp-pointed apical portion have also been visualized in tepals of species of Nymphaeaceae (Zini et al. 2017).Senescence of the sharppointed apical portion was also reported by Carpenter (2006) for four species of Nymphaeaceae.The sharp-pointed apical portion of hydropotes could have an important role in the physical protection of young leaves since, as with non-glandular trich mes (Tozin et al. 2016), they could provide a dense covering on the abaxial leaf surface as a defense for herbivory (Levin 1973;Baur et al. 1991).They may also facilitate the formation of film of air influencing its fluctuation (Barthlott et al. 2009).However, the main function of hydropotes changes later in development.

The general appearance of hydropotes with a sharppointed apical portion is similar to the mucilage hairs present in Cabombaceae (Carpenter 2006).However, the histochemical nature of the secretion produced by the hairlike portion of hydropotes of species of Nymphaeaceae has yet to be investigated.

Mature hydropotes of species of Nymphaea have been described as containing four cells (Lüttge 1964;Lüttge & Krapf 1969;Fahn 1979).However, the present study found them as comprising only three cells, which is in accordance with Carpenter (2006).We believe that the "cap cell", described by Lüttge (1964) and Lüttge & Krapf (1969) as above the lens-shaped cell, is not a proper cell, but instead debris from the detachment (see Fig. 3A-B) of the sharp-pointed apical portion, since a hair-like portion was not observed by these authors.

We found that the lens-shaped cell and the bowl-shaped cell of the studied hydropotes possess labyrinthine walls and an abundance of plasmodesmata, which are typical characteristics of cells involved in intense transport of substances (Lüttge & Krapf 1969).These features corroborate a role of hydropotes in the transport of water and minerals into and out of the plant (Fahn 1979 and references therein;Mishra & Dubey 2010), and are likely related to the bioremediation importance of hydropotes (Mishra & Dubey 2010).In a study involving light and scanning electron microscopy, Carpenter (2006) suggested that the darker color within the lens-shaped and bowl-shaped cells could be due to the presence of vesicles or crystals.Our analysis using transmission electron microscopy demonstrated that this denser aspect of the lens-shaped cell is likely due to the extensive ingrowths formed in the outer periclinal wall, in addition to the electron-dense cytoplasm.The occurrence of labyrinthine projections in cell walls has been reported for a variety of secretory structures (Bourett et al. 1994;Gama et al. 2016;Tozin & Rodrigues 2017), including hydropotes (Lüttge & Krapf 1969), and highlights the role of the cells bearing them in the transport of substances.Labyrinthine projections of alls increase the surface area of the plasma membrane supporting exchange among cells (Pate & Gunning 1972).In addition, the bowl-shaped cell possesses numerous mitochondria with dilated cristae, evidencing that this cell plays a role in generating energy (Evert 2006).The close physical juxtaposition between mitochondrial membranes in these cells was a remarkable feature and could optimize the movement of signaling molecules and the exchange of metabolites (Machado et al. 2018) involved in the hydropote functioning.The foot cell possesses plastids with starch grains and signs of degradation, indicating the importance of this cell in the storage of products of photosynthesis and supplying energy for the influx of water and mineral salts (Evert 2006;Taiz et al. 2017); the plastoglobuli also present in these plastids may play a key role in regulating photosynthesis regulation, plastid biogenesis and metabolism of various su stances (Wijk & Kessles 2017); and a wide central vacuole is typical of cells that are accumulating material (Evert 2006).Thus, we suggest functional compartmentalization among the three cells of mature hydropotes.

The path traveled by a solution, formed by water and mineral salts, seems to vary along the body of the hydropote.Influx from the environment to the lens-shaped cell seems be facilitated by the labyrinthine projections in the cell wa ls that enlarges the plasmalemma surface favoring the flow of solutions between the apoplast and the symplast (Pate & Gunning 1972).The influx from the lens-shaped cell to the bowl-shaped cell probably occurs via symplast through the numerous plasmodesmata connecting both cells.The abundance of mitochondria with dilated cristae in these cells suggests a high expenditure of energy is involved in this process.From bowl-shaped cell, the influx seems to continue due to, again, the labyrinthine projections in species of Nymphaea.In addition, the fusion of vesicles to the plasma