Anatomy and Histochemistry of the Roots and Shoots in the Aquatic Selenium Hyperaccumulator Cardamine Hupingshanensis (Brassicaceae)

Abstract The perennial selenium (Se) hyperaccumulator Cardamine hupingshanensis (Brassicaceae) thrives in aquatic and subaquatic Se-rich environments along the Wuling Mountains, China. Using bright-field and epifluorescence microscopy, the present study determined the anatomical structures and histochemical features that allow this species to survive in Se-rich aquatic environments. The roots of C. hupingshanensis have an endodermis with Casparian walls, suberin lamellae, and lignified secondary cell walls; the cortex and hypodermal walls have phi (Φ) thickenings; and the mature taproots have a secondary structure with a periderm. The stems possess a lignified sclerenchymal ring and an endodermis, and the pith and cortex walls have polysaccharide-rich collenchyma. Air spaces are present in the intercellular spaces and aerenchyma in the cortex and pith of the roots and shoots. The dense fine roots with lignified Φ thickenings and polysaccharide-rich collenchyma in the shoots may allow C. hupingshanensis to hyperaccumulate Se. Overall, our study elucidated the anatomical features that permit C. hupingshanensis to thrive in Se-rich aquatic environments.


Materials
The C. hupingshanensis samples were collected from the Hupingshan National Natural Reserve in Hunan Province, and from regions of the Yutangba and Liziping in Hubei Province along the Wuling Mountains, China. One-hundred twenty collected samples of C. hupingshanensis were preserved in the germplasm resource center of the Hubei Selenium Industry Technology Research Institute, China. Five mature specimens exhibiting normal growth from each collected site, namely Hupingshan, Yutangba, and Liziping, were observed under the microscope. The freshly sampled roots and shoots were fixed in formaldehyde-alcohol-acetic acid (FAA) [43] following collection. After fixing the tissues, freehand sections were cut using a two-sided blade. The sections were made at 5 mm, 15 mm, 30 mm, and 50 mm from the tip to the base with the cortex sloughed off (Fig. 1); through the middle of the stem and the base internode; and through the middle stem petioles and leaves. The sections were about 10 to 25 μm thick.
Little information exists on the anatomical and histochemical features of confirmed Se accumulators across various families, including Amaranthaceae, Asteraceae, Brassicaceae, Fabaceae, Rubiaceae, and Orobanchaceae [1,10]. While various biochemical and physiological analyses have confirmed that C. hupingshanensis hyperaccumulates Se and Cd [5,42], the associated structural and histochemical features of this species are yet to be elucidated. Accordingly, in this study we focused on determining the anatomical features of the roots and shoots of wild-type C. hupingshanensis that enable it to hyperaccumulate Se and survive its aquatic lifestyle. walls have obvious Φ thickenings in the 30 mm section. Narrow intercellular spaces can be observed between the cortex and hypodermis ( Fig. 2A-F).
The stele has a few secondary xylems; and the endodermis has obvious and lignified Casparian bands and is heavily suberized at the root base (Fig. 2G, H, I). The inner and radial cortex and hypodermal walls possess both large and small lignified Φ thickenings (Fig. 2G, I); the cortex and hypodermis are partially sloughed off; and the periderm appears suberized (Fig. 2H, I).

Taproots
The stele possesses diarch protoxylem poles; the endodermis has faint Casparian bands and almost complete suberin lamellae with a few passage cells; and the cortex and hypodermal walls have slightly lignified Φ thickenings at 5 mm from the root tip ( Fig. 3A, B). The endodermis has almost complete suberin lamellae in the 15 mm section (Fig. 3C).
The stele has a metaxylem and few secondary xylems; the endodermis has Casparian bands and lignin, becoming heavily suberized (Fig. 3D, E, F); and the inner and radial cortex walls have obviously suberized and lignified Φ thickenings at 30 mm. Intercellular spaces and aerenchyma can be observed between the cortex and hypodermis ( Fig. 3A-F).
The stele has a secondary xylem; the endodermis has lignified Casparian bands and is heavily suberized at 50 mm ( Fig. 4A, B, C). The inner and radial cortex and hypodermal walls possess both large and small lignified Φ thickenings; the cortex and hypodermis have been partially sloughed off; and the periderm is suberized and lignified ( Fig. 4A, B, C). The cortex and hypodermis have been sloughed off in the mature taproots; the stele has a secondary xylem and spacious parenchyma; and the periderm has suberized and lignified Casparian bands Here we demonstrated that the fine adventitious roots and primary structure of the taproots in the aquatic Se hyperaccumulator C. hupingshanensis exhibit similar anatomical and histochemical features. The roots have an endodermis and hypodermis with large cells. The cortex and hypodermal walls have lignified Φ thickenings that are greater near the endodermis. The taproot cortex has more cell layers than the fine adventitious roots, and the mature taproots have a secondary structure containing a periderm, as commonly observed in eudicots.
The young roots of C. hupingshanensis are similar in structure to another Se accumulator, O. javanica [6,32]. sections were stained with Sudan red 7B (SR7B) for the suberin lamellae [31,44], phloroglucinol-HCl (Pg) for lignin [43], berberine hemisulfate-aniline blue (BAB) for the Casparian bands and thickened cell walls [31,[45][46], and toluidine blue O (TBO) for the other structures including polysaccharides [31,37]. The specimens were examined using bright-field microscopy on a Leica DME microscope and photographed with a digital camera (Nikon E5400, Japan). Specimens stained with BAB were viewed under ultraviolet light on an Olympus IX71 epifluorescence microscope and photographed with a digital camera (RZ200C-21, China).

General morphology
Morphologically, C. hupingshanensis is characterized by erect stems (Fig. 1A) and taproots with a mass of fine adventitious roots (Fig. 1B). The fine adventitious roots contain one or two cell cortex layers (Fig. 2). The thick taproots possess three cell cortex layers in the primary structure (Fig. 3), with the cortex sloughed off in the secondary structure ( Fig. 1C; Fig. 4).
The fine adventitious roots and young taproots possess a diarch stele with a differentiating proto-and metaxylem; a cortex with an endodermis; and an enlarged outer ring with a distinct hypodermis. The cortex and hypodermal walls have lignified Φ thickenings. Mature taproots possess a typical secondary structure with a periderm.
The stems possess a lignified sclerenchymal ring enclosed within a central cylinder with scattered vascular bundles internal to the cortex, which possesses an endodermis. The pith and cortex walls contain polysaccharide-rich collenchyma. Aerenchyma and intercellular spaces are present in the cortex and pith of the roots and shoots.
The Φ thickenings of the roots of C. hupingshanensis are similar to those of some other brassicaceous species, The hypodermis of O. javanica has more cell layers and a cortex with spacious aerenchyma, though the cortex lacks lignified walls and Φ thickenings. Around the roots of O. javanica there are aerenchyma, and the walls possess suberin lamellae.

Stems and leaves
The stems possess a thickened lignified sclerenchymal ring enclosed within a central cylinder with scattered vascular bundles internal to the cortex. The sclerenchymal ring generally has vascular bundles inside it, and a spacious pith is present in the center of the sclerenchymal ring (Fig.  5A, B, C). The cortex has an endodermis with Casparian bands (Fig. 5D, E) and suberin (Fig. 5F) and lignin (Fig.  5C). The outer surface has a cuticle that reaches the inside of the epidermis (Fig. 5D, F). The pith and cortex have aerenchyma, and the walls have unlignified collenchyma that contain polysaccharides (Fig. 5A, B, C, D, F; see also [34]). Beneath the epidermis in the mature stems there is a peripheral mechanical ring (Fig. 5F).
The petioles possess one large and four small vascular bundles with lignified sclerenchymal rings and a spacious cortex with collenchyma and aerenchyma (Fig. 6A, B, C).
including B. oleracea and B. napus, and act as a barrier to ion transport [34][35]. Pelargonium hortorum has larger Φ thickenings at the hypodermis, which is opposite to what is observed in C. hupingshanensis [37]. Myrica rubra, P. malus, and G. biloba possess Φ thickenings near the endodermal radial walls but lack lignified walls [33,36,38]. Organelle-rich cytoplasm is present in the roots of the nickel (Ni) hyperaccumulator Senecio coronatus [11][12][13], and a Cd hyperaccumulating Arabidopsis thaliana genotype was found to possess dense root hairs [14]. We believe that lignified Φ thickenings in the roots might trap Se ions and contribute to the Se hyperaccumulation of C. hupingshanensis. The dense fine roots and lignified Φ thickenings may allow C. hupingshanensis to hyperaccumulate Se in a manner that differs from the Ni hyperaccumulator S. coronatus and the Cd hyperaccumulating A. thaliana genotype [11][12]14].
The role of air spaces in plant organs is to retain oxygen under hypoxic and anoxic conditions in order to enhance survival [18-19, 21-23, 26]. The fine roots and primary root structures of the taproots of C. hupingshanensis possess fewer intercellular air spaces and aerenchyma than O. javanica, P. distichum, P. arundinacea, A.   A cuticle is present on the surface (Fig. 6A). Cross-sections of the leaf blade reveals vascular bundles, palisade tissue, a cuticle, and vascular bundles that are slightly lignified (Fig. 6D, E, F).
The petioles of H. sibthorpioides and R. trichophyllus have an endodermis, but lack the lignified sclerenchymal ring around the vascular bundles and the cortex with collenchyma that are present in C. hupingshanensis [40][41]. The leaf blade structure of C. hupingshanensis is common to eudicots. We speculate that the polysaccharide-rich collenchyma walls of the pith and cortex in the shoots might enhance the tolerance of C. hupingshanensis to Se stress.
The cortex and pith of the stems of C. hupingshanensis possess a few aerenchyma lacunae, which is similar to that observed in H. sibthorpioides following submersion [41]. In contrast, H. altissima, P. distichum, A. lavandulaefolia, and A. selengensis have spacious pith cavities that might facilitate survival in heavily submerged conditions [28-30, 40, 47]. The stems of C. hupingshanensis are similar to those of the typical wetland-and aquatic-adapted plants H. sibthorpioides and R. trichophyllus, which possess an endodermis [40][41].

Conclusion
In summary, C. hupingshanensis possesses apoplastic barriers consisting of an endodermis, lignified Φ thickenings, and a cuticle, which is consistent with what has been found in studies on the effects of water stress on oxygen loss and solute transport in plants [18-19, 22-25, 32, 41, 45]. The lignified Φ thickenings in the roots and polysaccharide-rich collenchyma in the shoots might have evolved as key structural and histochemical features of