Apoplastic histochemical features of plant root walls that may facilitate ion uptake and retention

Abstract We used brightfield and epifluorescence microscopy, as well as permeability tests, to investigate the apoplastic histochemical features of plant roots associated with ion hyperaccumulation, invasion, and tolerance of oligotrophic conditions. In hyperaccumulator species with a hypodermis (exodermis absent), ions penetrated the root apex, including the root cap. By contrast, in non-hyperaccumulator species possessing an exodermis, ions did not penetrate the root cap. In vivo, the lignified hypodermis blocked the entry of ions into the cortex, while root exodermis absorbed ions and restricted them to the cortex. The roots of the hyperaccumulators Pteris vittata and Cardamine hupingshanensis, as well as the aquatic invasives Alternanthera philoxeroides, Eichhornia crassipes, and Pistia stratiotes, contained lignin and pectins. These compounds may trap and store ions before hypodermis maturation, facilitating ion hyperaccumulation and retention in the apoplastic spaces of the roots. These apoplastic histochemical features were consistent with certain species-specific characters, including ion hyperaccumulation, invasive behaviors in aquatic environments, or tolerance of oligotrophic conditions. We suggest that apoplastic histochemical features of the root may act as invasion mechanisms, allowing these invasive aquatic plants to outcompete indigenous plants for ions.

In this study, we aimed to identify the apoplastic histochemical features of the root cortical walls that facilitate ion uptake and retention, leading to ion hyperaccumulation and reflecting an adaptation to nutrient-deprived environments. To identify these features, we investigated the roots of seven representative hyperaccumulator, invasive, and/or oligotrophic plants: the aerial species, Pteris vittata and Chlorophytum comosum; the wetland species, Cardamine hupingshanensis and Paspalum distichum; and the aquatic species, Alternanthera philoxeroides, Eichhornia crassipes, and Pistia stratiotes. We also tested the apoplastic permeability of Pteris vittata and Paspalum distichum. An improved understanding of these plant roots' apoplastic histochemical features might help explain how these plants become invasive, tolerate oligotrophic conditions, and hyperaccumulate ions [4,5,8,[10][11][12]19,20,22,23,25,26]. These data will support the development of plants that can be used for the phytoremediation of ion-contaminated soils and oligotrophic water. Our results will also provide suggestions for the breeding of crops that can outcompete weed species [3,8,11,12,14,19,23,25,26].

Plant sourcing and collection
Mature specimens of Pteris vittata, Paspalum distichum, Chlorophytum comosum, Cardamine hupingshanensis, Alternanthera philoxeroides, Eichhornia crassipes, and Pistia stratiotes were identified in the Testing Ground of Yangtze University (Jingzhou City, Hubei Province, China) in October 2020. We collected samples of the adventitious aerial roots of Pteris vittata, which grow on walls in the cracks between bricks, and of Chlorophytum comosum, which propagate via shoots with adventitious aerial roots. We collected the roots of Cardamine hupingshanensis and Paspalum distichum from a wetland area. We collected the roots of Alternanthera philoxeroides, Eichhornia crassipes, and Pistia stratiotes from ponds. Ten roots were collected from each species of five plants and immediately fixed in formaldehyde-alcohol-acetic acid [57]. Eight fresh, intact specimens of Pteris vittata and Paspalum distichum were used for the apoplastic permeability tests [18,30,[33][34][35]].

Microstructure and histochemistry
Root tissues were sectioned freehand, using a two-sided razor blade, under a stereoscope (JNOEC JSZ6, China). Root sections were cut at 10 and 20 mm from the root tip, as well as at the point where the cortex began to slough off. Sections were divided into three sets, such that each set included sections of each plant and at same distance from the root tip. Each set of sections was then stained with one of three stains: 0.1% (w/v) berberine hemisulfate-aniline blue (BAB) to test for Casparian bands and lignin in the cell walls [38,58], phloroglucinol-HCl to test for lignin in the cell walls [59], and 0.02% (w/v) ruthenium red to test for pectin in the cell walls [55,60].
All sections were washed 2-3 times with sterile water, mounted with sterile water, and examined using brightfield microscopy under a Leica DME microscope (Germany). Specimens were photographed with a digital camera and a micrometer (Nikon E5400, Japan). Specimens stained with BAB were viewed under ultraviolet light on an Olympus IX71 epifluorescence microscope with excitation filter G 365 nm, absorption filter barriers U-WB (blue light), dichromatic mirror DM 500, compensation excitation filter BP 450-480, and compensation absorption filter BA 515. BABstained specimens were photographed using a digital camera and a micrometer (RZ200C-21, Ruizhi Cop., China) [27].

Apoplastic permeability
We tested the apoplastic permeability of whole fresh specimens of Pteris vittata and Paspalum distichum. We tested ion uptake using the apoplastic permeability tests of Seago et al., Meyer et al., and Meyer and Peterson [38,61,62], with modifications. In brief, we immersed the roots of the whole plants in the berberine solution without separating the roots from the plants; the plants remained intact. This modification allowed us to use the permeability tests to assess how the plants absorbed ions. Three intact plant roots were left unstained as the negative control. Three additional intact plants roots (tracer control) were immersed in 100 mL of 0.05% berberine hemisulfate for 1 h and washed with sterile water. The final three intact plant roots were immersed in 100 mL of 0.05% berberine hemisulfate for 1 h, washed with sterile water, immersed in 0.05 M potassium thiocyanate for 0.5 h, and washed again with sterile water. Roots were sectioned freehand and viewed under UV light as described by Seago et al. [38].

Results and discussion
At 10 mm from the tips of the adventitious aerial roots of Pteris vittata, the root wall contained pectins from the endodermis to the rhizodermis and hairs ( Figure 1a); the inner cortex had lignin-rich sclerenchyma layers and retained berberine around the endodermis ( Figure 1b); and the surfaces of the rhizodermis and hairs accumulated substantial amounts of berberine or berberine thiocyanate crystals (Figure 1b-d). Berberine penetrated to the cortex of the Pteris vittata roots close to the root tips ( Figure 1c and d), as indicated by the intense yellow fluorescence from the rhizodermis to the cortex. Similarly, intense yellow fluorescence was observed close to the tips of the roots of Paspalum distichum (Figure 1e), but berberine did not penetrate the root cap of this species. The walls of the adventitious aerial roots of Chlorophytum comosum also contained pectins from the endodermis to the rhizodermis and hairs (Figure 1f). Similar to Pteris vittata, the surfaces of the rhizodermis and hairs accumulated large amounts of berberine before metaxylem development (Figure 1g). After metaxylem development, the hairs were nearly sloughed off, but the exodermis and the rhizodermis surface continued to retain berberine (Figure 1h).
Before the cortex sloughed off, the adventitious roots of Cardamine hupingshanensis had pectins and lignin with even and Φ thickenings from the endodermis to the rhizodermis walls (Figure 2a-c). Similarly, pectins and lignified even thickenings were found from the endodermis to the rhizodermis walls in the adventitious roots of the aquatic plants Alternanthera philoxeroides (Figure 2d (Figure 3a and c).
In the apoplastic permeability test, the berberine tracer penetrated to the cortex of both Pteris vittata (exodermis absent) and Paspalum distichum (exodermis present) near the root tips [27,75], similar to what has been shown in Iris germanica (exodermis present) [61,86]. The berberine tracer also penetrated the root caps of Pteris vittata, similar to the results in Vicia faba (exodermis absent) [86]. However, the berberine tracer was unable to penetrate the root cap of Paspalum distichum, similar to what has been shown in Zea mays (exodermis present) and Iris germanica (exodermis present) [61,86]. Many berberine thiocyanate grains adhered to the mature hypodermis of Pteris vittata. By contrast, few berberine thiocyanate grains adhered to the mature exodermis of Paspalum distichum at the root surface [27,75]. The lignified hypodermis of Alternanthera philoxeroides blocks the entrance of ions into the cortex [18]. The root exodermis has only been shown to absorb berberine in vivo in Phalaris arundinacea, Zizania latifolia, and Artemisia spp. [30,34,35].

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.