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Root hair abundance impacts cadmium accumulation in Arabidopsis thaliana shoots

Root hair abundance impacts cadmium accumulation in Arabidopsis thaliana shoots Abstract Background and Aims Root hairs increase the contact area of roots with soil and thereby enhance the capacity for solute uptake. The strict hair/non-hair pattern of Arabidopsis thaliana can change with nutrient deficiency or exposure to toxic elements, which modify root hair density. The effects of root hair density on cadmium (Cd) accumulation in shoots of arabidopsis genotypes with altered root hair development and patterning were studied. Methods Arabidopsis mutants that are unable to develop root hairs (rhd6-1 and cpc/try) or produce hairy roots (wer/myb23) were compared with the ecotype Columbia (Col-0). Plants were cultivated on nutrient agar for 2 weeks with or without Cd. Cadmium was applied as Cd(NO3)2 at two concentrations, 10 and 100 µm. Shoot biomass, root characteristics (primary root length, lateral root number, lateral root length and root hair density) and Cd concentrations in shoots were assessed. Anatomical features (suberization of the endodermis and development of the xylem) that might influence Cd uptake and translocation were also examined. Key Results Cadmium inhibited plant growth and reduced root length and the number of lateral roots and root hairs per plant. Suberin lamellae in the root endodermis and xylem differentiation developed closer to the root apex in plants exposed to 100 µm Cd. The latter effect was genotype dependent. Shoot Cd accumulation was correlated with root hair abundance when plants were grown in the presence of 10 µm Cd, but not when grown in the presence of 100 µm Cd, in which treatment the development of suberin lamellae closer to the root tip appeared to restrict Cd accumulation in shoots. Conclusions Root hair density can have a large effect on Cd accumulation in shoots, suggesting that the symplasmic pathway might play a significant role in the uptake and accumulation of this toxic element. Arabidopsis thaliana, apoplasmic transport, symplasmic transport, cadmium (Cd), endodermis, root hair, mutant (rhd6-1, cpc/try, wer/myb23) INTRODUCTION Root hairs develop from specialized epidermal cells (trichoblasts) as tubular protrusions perpendicular to the root surface. Differences in root hair formation among species, and also the effects of the environment on their presence, abundance and length, have been known for many decades (von Guttenberg, 1968; Brown et al., 2017). In some species, root hairs can be formed from all epidermal (rhizodermal) cells, but most frequent is a pattern of root hair-forming and non-forming rhizodermal cells, termed ‘trichoblasts’ and ‘atrichoblasts’, respectively (Leavitt, 1904). In recent years, research on root hairs has focused largely on Arabidopsis thaliana, a Brassicales species with a distinct pattern of trichoblasts and atrichoblasts, arranged in alternating files along the root surface. The specification of trichoblasts depends on positional information and is executed through antagonistically acting transcription factor complexes (Berger et al., 1998; Dolan and Costa, 2001; Schiefelbein et al., 2009; Wang et al., 2010; Velasquez et al., 2016). Trichoblasts are located outside an anticlinal cortical cell wall, called the ‘H position’, facing the intercellular space between two underlying cortical cells, whereas atrichoblasts are located outside a periclinal cortical cell wall, called the ‘N position’, present over a single cortical cell. Young primary roots of arabidopsis possess eight files of cortical cells, thus there are eight root hair cell files, and from ten to 14 non-hair cell files (Dolan et al., 1994). However, the root meristem of arabidopsis (as in many other species) is dynamic, and changes as the root ages and the number of cell layers and files in the cortex and vascular cylinder changes (Baum et al., 2002). Root hairs are formed in the files from small, cytoplasmic cells (Dolan et al., 1993, 1994). These small cells are derived from initial cells, and many aspects of the genetic control of their development and mechanism of their formation are known (Grierson et al., 2001, 2014). Mutations of components of the activator complex, such as WER and MYB23, lead to root hair development in most rhizodermal cells, and mutations of components of the inhibitor complex, such as CPC and TRY, abolish the development of root hairs. Tip growth of root hairs during their elongation represents a unique form of plant cell growth, regulated by the cytoskeleton and accompanied by cell wall changes (Baluška et al., 2000; Ovečka et al., 2010). Both root hair initiation and tip growth are regulated by multiple phytohormones, including auxin and ethylene (Balcerowicz et al., 2015). Thus, root hair determination and elongation growth are very sensitive to environmental conditions that perturb phytohormone concentrations and their distribution (Wang et al., 2008; Bahmani et al., 2016; Pečenková et al., 2017). Root hairs increase the contact area of roots with soil and thereby enhance the capacity for nutrient and water uptake. The surface area of the root can be increased many fold by the presence of root hairs (Dittmer, 1937). They are generally short-lived structures and their presence collocates with the maximal absorption of water and nutrients by the root (White, 2012). The length and abundance of root hairs are often correlated with greater uptake and accumulation of mineral elements by roots, especially those with limited mobility in soils, such as inorganic phosphate (Pi), Mn2+, Fe3+, Zn2+ and K+ (Peterson and Steves, 2000; Brown et al., 2013; White, 2013; Salazar-Henao et al., 2016). Phosphate uptake has been studied most intensively, and the calculations of Föhse et al. (1991) suggested that, in soils with low available P, the root hairs might take up 90 % of the P accumulated by a plant. Root hairs are also essential for the formation of the rhizosheath of soil adhering to roots that provides tolerance to a variety of abiotic stresses, including drought and nutrient stresses (Brown et al., 2017). The role of root hairs in the uptake of non-essential elements has been studied less often. However, it has been observed that exposure of arabidopsis to cadmium (Cd) increases root hair density (Bahmani et al., 2016). Cadmium accumulation in soils generally occurs as a result of human activities, including the mining and refining of metal ores and the application of municipal compost and sewage sludge to agricultural soils (White and Greenwood, 2013). Cadmium concentrations <3 μg g–1 dry soil are recommended for agricultural soils to limit Cd concentration in edible produce (White et al., 2012). However, local concentrations close to former mining sites can reach several tens or hundreds of milligrams Cd g–1 dry soil (Alloway, 1995; Banásová et al., 2006, 2008). The availability of mutants with altered root hair initiation or development offers opportunities for studying the effects of root hair abundance on the uptake and accumulation of mineral nutrients and other elements. In the present work, we examined the role of root hairs in Cd uptake by comparing arabidopsis ecotype Columbia (Col-0) with mutants that are unable to develop root hairs (rhd6-1 and cpc/try) or in which most rhizodermal cells develop into hairs (wer/myb23) grown in the presence and absence of Cd. We also examined the development of the endodermis and xylem in these genotypes to determine the effects of other root structures that could affect the uptake and translocation of Cd by plants (Schreiber, 2010; Lux et al., 2011; Barberon, 2017; White and Pongrac, 2017). MATERIALS AND METHODS In vitro cultivation of arabidopsis plants and experimental design Arabidopsis thaliana mutants, which are either completely unable to develop root hairs [rhd6-1 in the ecotype Wassilewskija (Ws)] (Masucci and Schiefelbein, 1994) or form randomly one or two root hairs on the whole primary root surface [cpc/try, from a cross between mutants in the Ws ecotype and the Landsberg erecta (Ler) ecotype; Schellmann et al., 2002], and a double mutant in which most rhizodermal cells develop into hairs (wer/myb23 in the ecotype Col-0) (Kang et al., 2009) were compared with the ecotype Col-0. Schellmann et al. (2002) showed that the trichoblast/atrichoblast ratio was not significantly different in Ws and Ler, and Stetter et al. (2015) found no difference in root hair density between Col-0 and Ws. In the text, rhd6-1 and cpc/try are termed hairless mutants. The ecotype Col-0 has a distinct pattern of alternating files of root hair-forming and non-forming rhizodermal cells, whilst root hair formation is perturbed in the mutants (Fig. 1). Seeds were washed using 0.5 % sodium hypochlorite for 5 min, rewashed three times in sterile distilled water and placed on agar-solidified, hormone-free MS medium (Murashige and Skoog, 1962) containing 1 % sucrose and either 0, 10 or 100 μm Cd applied as Cd(NO3)2. Petri dishes (diameter 90 mm) containing 20 mL of medium were prepared under aseptic conditions. The choice of concentrations was based on preliminary experiments selecting mild 10 μm Cd stress (Cd 10) with limited effect on plant growth and severe stress 100 μm Cd (Cd 100) reducing growth >50 %. In addition we referred to several previous publications with application of these concentrations to arabidopsis and several other species (e.g. Martinez-Peñalver, 2012; Lukačová et al., 2013). The pH of each MS medium was adjusted to 5.8 using HCl or NaOH. A segment of the agar was removed and 15 seeds were sown on the cut surface to avoid contact of shoots with the media. This also enabled the roots to grow into the medium and not on its surface, ensuring homogenous exposure of the rhizodermis and root hairs to the medium. The Petri dishes were oriented vertically and plants were cultivated for 14 days after sowing (DAS). Seeds were stratified 2 d at 4 °C in the dark. Subsequently, Petri dishes were transferred to a controlled-environment growth chamber with a 16/8 h light/dark photoperiod, temperature of 26/20 °C (day/night) and 200 µmol m–2 s–1 light intensity of photosynthetically active radiation (PAR). Five Petri dishes were prepared for each genotype and treatment to provide enough material for all analyses. The experiment was repeated three times. Fig. 1. View largeDownload slide Details of the surface of primary roots (A–D) and sections with root hairs (E–H) of four Arabidopsis thaliana genotypes grown for 14 d in agar containing Murashige and Skoog (MS) medium. The genotype Columbia (Col-0), which has a distinct pattern of alternating root hair-forming and non-forming rhizodermal cells was used as a control (A, E). In the mutant wer/myb23, most epidermal cells develop hairs (B, F), whereas mutants rhd6-1 and cpc/try are unable to form root hairs (C, D and G, H, respectively). Arrows indicate root hairs. Note the development of root hairs in the ‘H position’ facing the intercellular space between two underlying cortical cells in Col-0, whereas the mutant wer/myb23 forms root hairs in both the ‘H position’ and also outside a periclinal cortical cell wall, called the ‘N position’. Scale bars = 80 µm (A–D), 30 µm (E–H). Fig. 1. View largeDownload slide Details of the surface of primary roots (A–D) and sections with root hairs (E–H) of four Arabidopsis thaliana genotypes grown for 14 d in agar containing Murashige and Skoog (MS) medium. The genotype Columbia (Col-0), which has a distinct pattern of alternating root hair-forming and non-forming rhizodermal cells was used as a control (A, E). In the mutant wer/myb23, most epidermal cells develop hairs (B, F), whereas mutants rhd6-1 and cpc/try are unable to form root hairs (C, D and G, H, respectively). Arrows indicate root hairs. Note the development of root hairs in the ‘H position’ facing the intercellular space between two underlying cortical cells in Col-0, whereas the mutant wer/myb23 forms root hairs in both the ‘H position’ and also outside a periclinal cortical cell wall, called the ‘N position’. Scale bars = 80 µm (A–D), 30 µm (E–H). Determination of growth parameters Fourteen days after sowing, plants in the Petri dishes were photographed using a Nikon D90 camera and macro objective AF-S Micro Nikkor 60 mm. Then shoots of ten plants from each Petri dish were carefully excised, washed in distilled water, surface dried with tissue paper and their fresh weight determined. Excised shoots were dried in a VENTI-Line (VWR) drying oven with forced air circulation at a temperature 70 °C for 3 d, and the dry weights of shoots were determined. Primary root length, number of lateral roots and the total length of the root system were evaluated on images of plants using ImageJ software (NIH). Lateral roots were counted and measured if their length was longer than 5 mm. In total, 30 roots from each genotype and treatment were examined. Root hair density and length determination At the end of the experiment, the roots were carefully removed from partially dissolved agar created by increasing the agar temperature temporarily to 40 °C. Roots for anatomical observations were fixed with methanol (Zelko et al., 2012) and stored in a refrigerator for later observations. Root hair length and density vary along the root axis. Therefore, the number of root hairs was counted using a Leica stereomicroscope (Leica M165FC) in three regions of the root: the sub-apical part of the root (2–5 mm from the root tip), a 5 mm long segment in the central part of the root and a 5 mm long segment from the base of the root excluding the hypocotyl junction. The total number of root hairs was calculated as the mean of the three root segments (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated and the data are means of three independent experiments. Cadmium concentration in the shoots The concentration of Cd was determined in shoot tissues using atomic absorption spectrometry (AAS Perkin Elmer Model 1100, at 228.8 nm with deuterium background correction) in the Geoanalytical Laboratories of the Institute of Geomaterials, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. Ten shoots from each Petri dish were combined to get the minimum dry weight for Cd determination. The samples were double step digested in PTFE pressure vessels in a microwave (Anton Paar Multiwave 3000) using concentrated HNO3 and H2O2 at a pressure of 60 bar. Calibration standards were prepared from a stock solution of Cd(NO3)2 (CertiPUR, Merck, Darmstadt, Germany). Tissue Cd concentrations were validated using a certified reference material (CRM NCS DC 73350 Leaves of Poplar, China National Analysis Center for Iron and Steel, Beijing, China). All measurements were performed in triplicate. Analysis of endodermal suberization and xylem lignification Whole-mount samples of shoots stored in methanol were cleared and stained with Fluorol Yellow 088 to identify suberin lamellae (Brundrett et al., 1991; Lux et al., 2005, 2015) and with phloroglucinol-HCl to identify lignification of the xylem. The roots used to identify suberin lamellae were examined under a fluorescence microscope (Axioskop 2 plus, Carl Zeiss, Germany; filter set Carl Zeiss N. 25: excitation filter TBP 400 nm + 495 nm + 570 nm, chromatic beam splitter TFT 410 nm + 505 nm + 585 nm, and emission filter TBP 460 nm + 530 nm + 610 nm) and documented using a digital camera DP72 (Olympus). For measurements, Lucia G 4.80 (LIM, Czech Republic) software was used. The same microscope and camera were used for bright field and/or dark field observation and documentation. In each experiment, at least five primary roots were used for analyses. Statistical analysis Analysis of variance (ANOVA) was performed using Statgraphics Centurion XV.I statistical software. Statistical significance was defined at P <0.05. All data are expressed as the mean ± s.e. for a stated number of replications. All experiments were repeated three times. RESULTS Cadmium accumulation in shoots of wild-type and mutant plants differing in root hair abundance Cadmium concentrations in the shoots of all genotypes increased with increasing Cd concentration in the medium (Fig. 2). Hairy plants (wer/myb23) had greater Cd concentrations in their shoots, and hairless plants (rhd6-1 and cpc/try) had lower Cd concentrations in their shoots, than wild-type plants (Fig. 2). Cadmium concentrations in the shoots of wer/myb23 from the Cd 100 treatment reached 975 μg g–1 d. wt (Fig. 2). Shoots of the hairless mutants rhd6-1 and cpc/try had Cd concentrations of 635 and 636 μg g–1 d. wt, respectively, and shoots of Col-0 had a concentration of 765 μg g–1 d. wt when grown in the Cd 100 treatment. These differences were statistically significant between wer/myb23 and Col-0, and between the hairless mutants and Col-0. In the Cd 10 treatment, wer/myb23 had a shoot Cd concentration of 396 μg g–1 d. wt, whilst the hairless mutants, cpc/try and rhd6-1, had shoot Cd concentrations of 195 and 267 μg g–1 d. wt, respectively. The shoot Cd concentration of wer/myb23 was significantly greater than that of all genotypes, and the shoot Cd concentration of cpc/try was significantly lower than that of Col-0. The differences between Col-0 and rhd6-1 and between rhd6-1 and cpc/try were not significant in the Cd 10 treatment (Fig. 2). Fig. 2. View largeDownload slide Cadmium concentrations, expressed on a dry weight (d. wt) basis, in shoots of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into root hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P < 0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Fig. 2. View largeDownload slide Cadmium concentrations, expressed on a dry weight (d. wt) basis, in shoots of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into root hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P < 0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Growth parameters, root hair length and density Seedlings grown in the absence of Cd developed several leaves during the experiment (Fig. 3). Shoot growth was inhibited by the presence of Cd in the agar, and the magnitude of this inhibition depended on genotype and Cd concentration in the agar (Figs 3 and 4B; Supplementary Data Fig. S1B). Similarly, the length of primary roots was reduced in the presence of Cd, and the magnitude of this effect depended upon the genotype and Cd treatment (Figs 3 and 4A; Supplementary Data Fig. S1A). When plants were grown in the absence of Cd, Col-0 (4.13 cm) and cpc/try (4.29 cm) had the longest primary roots and wer/myb23 (3.61 cm) and rhd6-1 (3.73 cm) had the shortest primary roots. The difference between these two groups was significant (Fig. 4A). The Cd 10 treatment inhibited primary root growth significantly, except in wer/myb23. However, the absolute inhibition of primary root growth in all genotypes was small (Fig. 4A). On the other hand, the Cd 100 treatment resulted in a highly significant reduction in primary root length in all genotypes to approximately one-third of the values observed in the absence of Cd (Fig. 4A). Primary root lengths of Col-0, wer/myb23 and rhd6-1 (1.28, 1.30 and 1.17 cm, respectively) were not significantly different in plants from the Cd 100 treatments. The hairless mutant cpc/try had significantly longer primary roots than other genotypes in both Cd treatments (4.01 cm in Cd 10 and 1.54 cm in Cd 100). There was no positive correlation between primary root length and shoot Cd concentration. Shoot fresh weight was reduced by the presence of Cd in the medium (Fig. 4B). The shoot fresh weight of wer/myb23 was greater than that of other genotypes, whether assayed in the absence or presence of Cd (Fig. 4B). The Cd 10 treatment did not influence shoot fresh weight greatly. However, the Cd 100 treatment reduced shoot fresh weight significantly in all genotypes when compared with the control and the Cd 10 treatment. The reduction in shoot fresh weight was >50 % in all genotypes. Shoot dry weight was also reduced by the presence of Cd in the growth medium (Fig. 4C). Again, wer/myb23 had greater shoot dry weight than other genotypes when assayed under control or Cd 10 conditions (Fig. 4C). Shoot dry weight was least in rhd6-1, whether assayed in the presence or absence of Cd. The effect of the Cd 10 treatment on dry weight was greater than its effect on fresh weight. In the Cd 100 treatment, the shoot dry weight of cpc/try was greater than that of other genotypes. Fig. 3. View largeDownload slide Effects of cadmium on the growth of the ecotype Columbia (Col-0) (A, E, I) the mutant wer/myb23, in which most epidermal cells develop into hairs (B, F, J), and the mutants rhd6-1 (C, G, K) and cpc/try (D, H, L), which are unable to form root hairs. Seeds were placed on the surface of an agar slab placed within Petri dishes oriented vertically to enable shoots to develop without contact with agar and roots to grow within the agar. Plants were cultivated for 14 d in media containing no Cd (Control, C; A–D or either 10 μm Cd (Cd 10; E–H) or 100 μm (Cd 100; I–L) supplied as Cd(NO3)2. Scale bars = 1 cm. Fig. 3. View largeDownload slide Effects of cadmium on the growth of the ecotype Columbia (Col-0) (A, E, I) the mutant wer/myb23, in which most epidermal cells develop into hairs (B, F, J), and the mutants rhd6-1 (C, G, K) and cpc/try (D, H, L), which are unable to form root hairs. Seeds were placed on the surface of an agar slab placed within Petri dishes oriented vertically to enable shoots to develop without contact with agar and roots to grow within the agar. Plants were cultivated for 14 d in media containing no Cd (Control, C; A–D or either 10 μm Cd (Cd 10; E–H) or 100 μm (Cd 100; I–L) supplied as Cd(NO3)2. Scale bars = 1 cm. Fig. 4. View largeDownload slide The effects of cadmium on the (A) primary root length, (B) shoot fresh weight, (C) shoot dry weight, (D) number of lateral roots and (E) total root length of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P <0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Asterisks indicate significant differences between treatments within genotypes. Fig. 4. View largeDownload slide The effects of cadmium on the (A) primary root length, (B) shoot fresh weight, (C) shoot dry weight, (D) number of lateral roots and (E) total root length of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P <0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Asterisks indicate significant differences between treatments within genotypes. If Cd was taken up preferentially by root cells at the root tip or solely by cells on the surface of the root, then the number of root tips, i.e. the ramification of the root by formation of lateral roots and the total root length of individual genotypes, becomes important (Fig. 4D, E; Supplementary Data Fig. S1D, E). In control conditions, the highest number of laterals (approximately ten per plant) was in Col-0 and cpc/try, followed by rhd6-1 (approximately nine per plant) and wer/myb23 (approximately seven per plant). Even low Cd concentrations in the agar (Cd 10 treatment) reduced the number of lateral roots (Fig. 4D). This occurred in all genotypes, with the greatest decrease (approx. 60 %) being in cpc/try. The Cd 100 treatment had even more of an effect, with an approx. 80 % reduction in the number of lateral roots in Col-0 and a 70 % reduction in wer/myb23 and rhd6-1. In cpc/try, the number of lateral roots was similar in both the Cd 10 and Cd 100 treatments. In the absence of Cd, the total root length, i.e. the sum of the lengths of the primary and all lateral roots of a plant, was greater in rhd6-1 (9.7 cm) and Col-0 (9.3 cm) than in cpc/try (8.4 cm) or wer/myb23 (6.4 cm). The presence of Cd in the agar reduced the total root length of all genotypes. Col-0 had the greatest total root length in the Cd 10 treatment, and cpc/try had the least. In contrast, in the Cd 100 treatment, cpc/try had the greatest total root length and rhd6-1 had the least (Fig. 4E). The hairless mutants rhd6-1 and cpc/try had few root hairs (Fig. 1). In contrast, most rhizodermal cells developed into root hairs in the wer/myb23 mutant, which had about twice as many root hairs per plant as the Col-0 ecotype in all treatments (Table 1). The Cd 10 treatment did not influence the number of root hairs in the wer/myb23 mutant, but reduced the number of root hairs in Col-0 to about 78 % of the number observed in the absence of Cd. The Cd 100 treatment reduced the number of root hairs to about 34 % of their number in the absence of Cd in both wer/myb23 and Col-0 (Table 1). The number of root hairs per millimetre increased from 30 in the absence of Cd to 43 in the Cd 100 treatment in wer/myb23 and from nine in the absence of Cd to 20 in the Cd 100 treatment in Col-0. The presence of Cd, especially the 100 Cd treatment, induced the frequent formation of irregularly occurring, unusually shaped, balloon-like, bulbous or pear-like root hairs in both wer/myb23 and Col-0 (Fig. 5A, B). In the hairless cpc/try mutant, occasional swelling of rhizodermal cells occurred (Fig. 5C). Fig. 5. View largeDownload slide The effect of 100 μm Cd(NO3)2 on the morphology of the rhizodermis and root hairs of (A) the ecotype Col-0, (B) the wer/myb23 mutant and (C) the cpc/try mutant. Abnormally shaped, balloon-like root hairs (arrows) occurred in both Col-0 and wer/myb23. Swollen rhizodermal cells (arrowheads) occurred occasionally in cpc/try. Scale bars = 100 μm. Fig. 5. View largeDownload slide The effect of 100 μm Cd(NO3)2 on the morphology of the rhizodermis and root hairs of (A) the ecotype Col-0, (B) the wer/myb23 mutant and (C) the cpc/try mutant. Abnormally shaped, balloon-like root hairs (arrows) occurred in both Col-0 and wer/myb23. Swollen rhizodermal cells (arrowheads) occurred occasionally in cpc/try. Scale bars = 100 μm. Table 1. The effects of cadmium on the number of root hairs per plant of Arabidopsis thaliana ecotype Columbia (Col-0) and a mutant in which most epidermal cells develop into hairs (wer/myb23) Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. The total number of root hairs was calculated as the mean of the three root segments viewed (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated. Data are expressed as means ± s.e. from three independent experiments. View Large Table 1. The effects of cadmium on the number of root hairs per plant of Arabidopsis thaliana ecotype Columbia (Col-0) and a mutant in which most epidermal cells develop into hairs (wer/myb23) Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. The total number of root hairs was calculated as the mean of the three root segments viewed (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated. Data are expressed as means ± s.e. from three independent experiments. View Large Anatomical characteristics that might influence Cd uptake and translocation It has been proposed that Cd might reach the xylem, and thence the shoot, by either an apoplasmic or a symplasmic pathway across the root (Lux et al., 2011). The barrier to apoplasmic transport of solutes to the stele is at the endodermis (White, 2001; Lux et al., 2011; Barberon, 2017). The first stage in the development of the endodermis is the formation of Casparian bands, which is followed closely by the second stage in which suberin lamellae cover the whole inner surface of endodermal cells. The formation of the Casparian band restricts transport through the apoplast to the stele, and the suberization of endodermal cells prevents solute transport across the plasma membrane of endodermal cells, thereby ensuring symplasmic transport to the stele (White, 2001; Moore et al., 2002; Lux et al., 2011; Barberon, 2017). The rate of development of the endodermis, and its response to Cd in the rhizosphere, differs between Col-0, wer/myb23, rhd6-1 and cpc/try (Fig. 6). In the absence of Cd in the rhizosphere the distance of suberin lamellae from the root tip in Col-0 was similar to that in the hairless mutants rhd6-1 and cpc/try. Suberin deposition began about 2.8 mm from the root tip, and well-developed suberin lamellae were formed at about 3.3 mm from the root tip. In wer/myb23, these distances were about 1 mm further from the root tip (Figs 6 and 7). Development of the endodermis differed little between the control and the Cd 10 treatment, although suberization of the endodermis in wer/myb23 occurred closer to the root tip in the Cd 10 treatment than in the absence of Cd. The Cd 100 treatment resulted in the development of the endodermis closer to the root tip in all genotypes. In Col-0 and wer/myb23, suberization of the endodermal cells occurred 1–1.5 and 1.5–2 mm from the tip, respectively. In the hairless mutants, rhd6-1 and cpc/try, suberization of the endodermal cells occurred further away from the root tip, at 2.2–2.8 mm from the root tip in rhd6-1 and at 2.8–3.3 mm from the root tip in cpc/try (Figs 6 and 7). In these hairless mutants, irregular and patchy formation of suberin lamellae took place along an extended length of the root, which did not occur in Col-0 or wer/myb23. Fig. 6. View largeDownload slide Graphical summary of the effects of increasing agar Cd concentration on suberization of the endodermis and xylem development in roots of the Arabidopsis thaliana ecotype Columbia (Col-0), wer/myb23, a mutant in which most epidermal cells develop into hairs, and the mutants rhd6-1 and cpc/try that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C) and either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. The root anatomy of each genotype is shown in cross-section at the top of the figure according to the basic scheme of Salazar-Henao et al. (2016). The changing position of root hair initiation, onset of protoxylem differentiation (red line) and deposition of suberin lamellae (the beginning of suberin lamellae deposition is indicated by a dashed green line and complete suberin lamellae deposition by a solid green line) in the endodermis at the root apices of the four genotypes are shown in longitudinal views. Fig. 6. View largeDownload slide Graphical summary of the effects of increasing agar Cd concentration on suberization of the endodermis and xylem development in roots of the Arabidopsis thaliana ecotype Columbia (Col-0), wer/myb23, a mutant in which most epidermal cells develop into hairs, and the mutants rhd6-1 and cpc/try that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C) and either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. The root anatomy of each genotype is shown in cross-section at the top of the figure according to the basic scheme of Salazar-Henao et al. (2016). The changing position of root hair initiation, onset of protoxylem differentiation (red line) and deposition of suberin lamellae (the beginning of suberin lamellae deposition is indicated by a dashed green line and complete suberin lamellae deposition by a solid green line) in the endodermis at the root apices of the four genotypes are shown in longitudinal views. Fig. 7. View largeDownload slide Effects of increasing Cd concentration on the formation of root hairs and the development of suberin lamellae in the endodermis of the Arabidopsis thaliana ecotype Columbia (Col-0), (A, B, I, J); wer/myb23, a mutant in which most epidermal cells develop into hairs (C, D, K, L); and the mutants rhd6-1 (E, F, M, N) and cpc/try (G, H, O, P) that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C), (A, C, E, G) or 100 μm (Cd 100), (B, D, F, H) supplied as Cd(NO3)2. Arrows indicate the beginning of suberin lamellae deposition in the endodermis. (I–P) Detail of the root in which suberization of the endodermis has just begun. Note the patchy development of suberin lamellae (occurring in individual endodermal cells) in mutant rhd6-1 (N). Scale bars = 600 μm (A–H), 80 μm (I–P). Fig. 7. View largeDownload slide Effects of increasing Cd concentration on the formation of root hairs and the development of suberin lamellae in the endodermis of the Arabidopsis thaliana ecotype Columbia (Col-0), (A, B, I, J); wer/myb23, a mutant in which most epidermal cells develop into hairs (C, D, K, L); and the mutants rhd6-1 (E, F, M, N) and cpc/try (G, H, O, P) that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C), (A, C, E, G) or 100 μm (Cd 100), (B, D, F, H) supplied as Cd(NO3)2. Arrows indicate the beginning of suberin lamellae deposition in the endodermis. (I–P) Detail of the root in which suberization of the endodermis has just begun. Note the patchy development of suberin lamellae (occurring in individual endodermal cells) in mutant rhd6-1 (N). Scale bars = 600 μm (A–H), 80 μm (I–P). Development of xylem, with lignified cell walls of protoxylem developing in the periphery of the central cylinder, generally coincides with root hair development (Demchenko and Kalimova, 2008). In the hairless mutants rhd6-1 and cpc/try, development of the xylem started closer to the root tip than in Col-0 and the wer/myb23 mutant in the Cd 10 treatment. DISCUSSION It is the aim of many crop scientists to supply sufficient, nutritious food and fodder for the future (White et al., 2012). It is also essential that both food and fodder are safe to eat. Cadmium is toxic to all living organisms, and its presence, accumulation and effect on plants has been intensively studied. Research is focused on the development of crops that can tolerate Cd in the environment and accumulate less in their edible parts or to develop plants that can accumulate Cd for the phytoremediation of contaminated land (Prasad, 1995; Das et al., 1997; McGrath et al., 2001; Lux et al., 2004, 2011; Van Belleghem et al., 2007; Lukačová-Kuliková and Lux, 2010; Singh et al., 2011; Vaculík et al., 2012; White et al., 2012; Martinka et al., 2014; White and Pongrac, 2017). The present study shows that shoots of the hairy mutant, wer/myb23, had greater Cd concentrations than shoots of the ecotype Col-0 and that shoots of both wer/myb23 and Col-0 had greater Cd concentrations than shoots of the hairless mutants rhd6-1 and cpc/try (Fig. 2). The shoot Cd concentrations reported here are relatively high compared with previous studies of arabidopsis and other species exposed to Cd (Bovet et al., 2003; Lukačová-Kuliková and Lux, 2010). One reason for this might be that the availability of Cd is greater from agar than from soil substrates. All genotypes showed inhibition of root growth and reduced shoot biomass as a result of Cd treatment (Figs 3 and 4), which is consistent with the toxicity of Cd to plants (Prasad, 1995; Lux et al., 2011; White and Pongrac, 2017). However, genotypes differed in their root development and shoot biomass both in the absence and in the presence of Cd (Figs 3 and 4). The hairy mutant, wer/myb23, had a shorter primary root, formed fewer lateral roots and had a shorter total root length than Col-0 in the absence of Cd and in the Cd 10 treatment (Fig. 4A, D, E), but had a greater shoot fresh weight and dry weight under these conditions, as well as in the Cd 100 treatment (Fig. 4B, C). The hairless mutant, rhd6-1, although in ecotype Ws, behaved similarly to Col-0 in both root development and shoot biomass, whereas the hairless mutant cpc/try developed longer primary roots, more lateral roots and a greater total root length than other genotypes in the Cd 100 treatment. The hairless double mutant cpc/try had greater shoot fresh weight than Col-0 in the Cd 10 treatment and more shoot dry weight than Col-0 in the Cd 100 treatment. The total number of root hairs was about twice as great in the hairy mutant wer/myb23 than in Col-0 (Table 1). In both wer/myb23 and Col-0, root hairs developed closer to the root apex in the Cd 100 treatment (Fig. 6). Differences in the shoot phenotypes of the two hairless genotypes might occur because of their different genetic backgrounds, rhd6-1 being derived from ecotype Ws and cpc/try from a cross between mutants in Ws and Ler ecotypes. However, the data reveal no differences between the hairless genotypes in their shoot Cd concentrations (Fig. 2). Recent studies have indicated a significant contribution of root hairs to the uptake of some toxic elements. Zheng et al. (2011) concluded that root hairs contributed significantly to Cd uptake by barley, and Balestri et al. (2014) noted a possible involvement of root hairs in Cd uptake by Pteris vittata. In arabidopsis, the number and length of root hairs in the apical 5 mm of the root increased in the presence of Cd (Bahmani et al., 2016). This is consistent with the results obtained here for both Col-0 and wer/myb23. However, it should be noted that, due to the reduced total root length, the total number of root hairs per plant was reduced (Table 1). In the Cd 10 treatment, the Cd content of the shoot was strongly correlated with the number of root hairs per plant across all genotypes studied (Fig. 7), suggesting a contribution of root hairs to Cd uptake and the presence of a symplasmic pathway to the xylem under these conditions. However, the lack of a correlation between the Cd content of the shoot and the number of root hairs in the Cd 100 treatment suggests that both symplasmic and apoplasmic pathways contribute to Cd delivery to the xylem when the rhizosphere Cd concentration is large (Fig. 7). When arabidopsis roots are exposed to Cd, it accumulates in both the apoplasmic and symplasmic space (van Beleghem et al., 2007). Several studies have indicated the importance of the development of apoplasmic barriers in the endodermis and exodermis in restricting Cd uptake by roots and Cd accumulation in the shoot (Lux et al., 2004, 2011; Ďurčeková et al., 2007; Vaculík et al., 2009; Lukačová-Kuliková and Lux, 2010). Ontogenesis of both these highly specialized root layers is characterized by three stages: (1) the formation of Casparian bands close to the root tip; (2) the development of suberin lamellae around endodermal cells; and (3) the thickening of cell walls of endodermal cells (von Guttenberg, 1968; White, 2001; Schreiber, 2010; Barberon, 2017). Variation in the development of the endodermis has been observed among ecotypes of wild species and cultivars of crops, and correlated with Cd accumulation in the shoot (Lux et al., 2004; Lukačová-Kuliková and Lux, 2010; Vaculík et al., 2012; Tao et al., 2017). The translocation of Cd to the shoot is lower in genotypes with accelerated development of apoplasmic barriers, close to the root tip, when compared with genotypes developing these barriers more distant from the root tip. The development of the endodermis is extremely sensitive to environmental conditions (Redjala et al., 2011; Lux et al., 2011; Líška et al., 2016), and several studies have shown that abiotic stresses, including the presence of Cd in the rhizosphere, induce early formation of apoplasmic barriers by premature deposition of suberin and lignin to the cell walls (Reinhardt and Rost, 1995; Schreiber et al., 1999; Enstone et al., 2002; Lux et al., 2011; Balestri et al., 2014; Martinka et al., 2014; Líška et al., 2016). In a previous study, unilateral exposure of maize roots to Cd resulted in earlier development of apoplasmic barriers in the side of the root exposed to Cd (Lux et al., 2011). In the present experiments, it was observed that exposure of roots to the Cd 100 treatment resulted in suberin lamellae being formed closer to the root tip (Fig. 6). It is possible that this might restrict the uptake of Cd and Cd accumulation in shoots. This is consistent with the lower Cd content of shoots of Col-0 than of shoots of rhd6-1 and cps/try in the Cd 100 treatment (Fig. 7). However, it is inconsistent with the greater Cd content of shoots of wer/myb23 than shoots of rhd6-1 and cps/try in the Cd 100 treatment (Fig. 7). Thus, in addition to Cd movement to the xylem via an apoplasmic pathway, the involvement of a symplasmic pathway to the xylem facilitated by root hairs might be suggested even in the Cd 100 treatment. The irregular and patchy development of suberin lamellae in rhd6-1 and also the very early development of protoxylem close to the root tip in this genotype might also contribute to the differences in Cd accumulation in the shoot among the genotypes studied. Shorter hairs are formed in roots inside agar compared with longer and thin hairs of roots growing on the agar surface, developing without contact with Cd-contaminated agar only in the humid air (Fig. 8). Additionally, abnormal development of root hairs was observed in both Col-0 and wer/myb23 in the Cd 100 treatment, suggesting a direct effect of Cd on hair initiation and tip growth (Fig. 5). These processes are regulated by plant hormones, Ca channels at the apex of the hair and cytoskeletal rearrangements (Baluška et al., 2000; Ryan et al., 2001; Scheifelbein et al., 2014; White, 2015), and are sensitive to various environmental stresses (Mueller and Schmidt, 2004; Balcerowicz et al., 2015; Bahmani et al., 2016). Cell wall visco-elastic properties can also be affected by toxic elements (Ma et al., 2004; Krupinski et al., 2016). Fig. 8. View largeDownload slide Difference in development of root hairs (arrows) of Arabidopsis thaliana ecotype Columbia (Col-0) cultivated in vitro with roots growing (A) within agar-solidified MS medium (agar) or (B) on the surface of the same medium (air). Note the difference in the root hair length and thickness. Scale bars = 70 μm. Fig. 8. View largeDownload slide Difference in development of root hairs (arrows) of Arabidopsis thaliana ecotype Columbia (Col-0) cultivated in vitro with roots growing (A) within agar-solidified MS medium (agar) or (B) on the surface of the same medium (air). Note the difference in the root hair length and thickness. Scale bars = 70 μm. CONCLUSIONS Shoots of the hairy genotype, wer/myb23, had greater Cd concentrations, and shoots of the hairless mutants, rhd6-1 and cpc/try, had lower Cd concentrations, than shoots of the ecotype Col-0 in both Cd 10 and Cd 100 treatments. In the Cd 10 treatment, the Cd content of the shoot was strongly correlated with the number of root hairs per plant across all genotypes studied, suggesting a contribution of root hairs to Cd uptake and that a symplasmic pathway contributed to Cd movement to the xylem under these conditions. The Cd content of the shoot was weakly correlated with the number of root hairs in the Cd 100 treatment, suggesting that the symplasmic pathway contributed less to the movement of Cd to the xylem at greater rhizosphere Cd concentrations. It is suggested that the development of the endodermis closer to the root apex in Col-0 and wer/myb23 reduces the potential apoplasmic movement of Cd to the xylem in these genotypes relative to rhd6-1 and cpc/try, which form their endodermis at a greater distance from the root apex. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Figure S1: the effects of cadmium on the primary root length, shoot fresh weight, shoot dry weight, number of lateral roots and total root length of Arabidopsis thaliana genotypes Col-0, wer/myb23, rhd6-1 and cpc/try. Figure S2: correlation between the Cd content of the shoot and number of root hairs per plant. ACKNOWLEDGEMENTS This work was supported by the Slovak Research and Development Agency, contract No. APVV SK-AT-2015-0009, by the Slovak Grant Agency VEGA, grant no. VEGA 1/0605/17, the Austrian Science Fund ANR-FWF I 1725-B16 and the Scientific & Technological Cooperation (WTZ) Austria & Slovakia SK 04/2016. We thank Kai Dünser for providing rhd6-1, cpc/try and wer/myb23 seeds. The reviewers are thanked for their helpful comments. P.J.W. was supported by the Rural & Environment Science & Analytical Services Division of the Scottish Government. This paper as a part of Special Issue is dedicated to Peter Barlow, our dear colleague and friend. LITERATURE CITED Alloway BL . 1995 . Heavy metals in moils . 2nd edn . Glasgow, UK : Blackie Academic and Professional an imprint of Chapman and Hall . Google Scholar Crossref Search ADS Bahmani R , Kim DG , Kim JA , Hwang S . 2016 . 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Root hair abundance impacts cadmium accumulation in Arabidopsis thaliana shoots

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Abstract Background and Aims Root hairs increase the contact area of roots with soil and thereby enhance the capacity for solute uptake. The strict hair/non-hair pattern of Arabidopsis thaliana can change with nutrient deficiency or exposure to toxic elements, which modify root hair density. The effects of root hair density on cadmium (Cd) accumulation in shoots of arabidopsis genotypes with altered root hair development and patterning were studied. Methods Arabidopsis mutants that are unable to develop root hairs (rhd6-1 and cpc/try) or produce hairy roots (wer/myb23) were compared with the ecotype Columbia (Col-0). Plants were cultivated on nutrient agar for 2 weeks with or without Cd. Cadmium was applied as Cd(NO3)2 at two concentrations, 10 and 100 µm. Shoot biomass, root characteristics (primary root length, lateral root number, lateral root length and root hair density) and Cd concentrations in shoots were assessed. Anatomical features (suberization of the endodermis and development of the xylem) that might influence Cd uptake and translocation were also examined. Key Results Cadmium inhibited plant growth and reduced root length and the number of lateral roots and root hairs per plant. Suberin lamellae in the root endodermis and xylem differentiation developed closer to the root apex in plants exposed to 100 µm Cd. The latter effect was genotype dependent. Shoot Cd accumulation was correlated with root hair abundance when plants were grown in the presence of 10 µm Cd, but not when grown in the presence of 100 µm Cd, in which treatment the development of suberin lamellae closer to the root tip appeared to restrict Cd accumulation in shoots. Conclusions Root hair density can have a large effect on Cd accumulation in shoots, suggesting that the symplasmic pathway might play a significant role in the uptake and accumulation of this toxic element. Arabidopsis thaliana, apoplasmic transport, symplasmic transport, cadmium (Cd), endodermis, root hair, mutant (rhd6-1, cpc/try, wer/myb23) INTRODUCTION Root hairs develop from specialized epidermal cells (trichoblasts) as tubular protrusions perpendicular to the root surface. Differences in root hair formation among species, and also the effects of the environment on their presence, abundance and length, have been known for many decades (von Guttenberg, 1968; Brown et al., 2017). In some species, root hairs can be formed from all epidermal (rhizodermal) cells, but most frequent is a pattern of root hair-forming and non-forming rhizodermal cells, termed ‘trichoblasts’ and ‘atrichoblasts’, respectively (Leavitt, 1904). In recent years, research on root hairs has focused largely on Arabidopsis thaliana, a Brassicales species with a distinct pattern of trichoblasts and atrichoblasts, arranged in alternating files along the root surface. The specification of trichoblasts depends on positional information and is executed through antagonistically acting transcription factor complexes (Berger et al., 1998; Dolan and Costa, 2001; Schiefelbein et al., 2009; Wang et al., 2010; Velasquez et al., 2016). Trichoblasts are located outside an anticlinal cortical cell wall, called the ‘H position’, facing the intercellular space between two underlying cortical cells, whereas atrichoblasts are located outside a periclinal cortical cell wall, called the ‘N position’, present over a single cortical cell. Young primary roots of arabidopsis possess eight files of cortical cells, thus there are eight root hair cell files, and from ten to 14 non-hair cell files (Dolan et al., 1994). However, the root meristem of arabidopsis (as in many other species) is dynamic, and changes as the root ages and the number of cell layers and files in the cortex and vascular cylinder changes (Baum et al., 2002). Root hairs are formed in the files from small, cytoplasmic cells (Dolan et al., 1993, 1994). These small cells are derived from initial cells, and many aspects of the genetic control of their development and mechanism of their formation are known (Grierson et al., 2001, 2014). Mutations of components of the activator complex, such as WER and MYB23, lead to root hair development in most rhizodermal cells, and mutations of components of the inhibitor complex, such as CPC and TRY, abolish the development of root hairs. Tip growth of root hairs during their elongation represents a unique form of plant cell growth, regulated by the cytoskeleton and accompanied by cell wall changes (Baluška et al., 2000; Ovečka et al., 2010). Both root hair initiation and tip growth are regulated by multiple phytohormones, including auxin and ethylene (Balcerowicz et al., 2015). Thus, root hair determination and elongation growth are very sensitive to environmental conditions that perturb phytohormone concentrations and their distribution (Wang et al., 2008; Bahmani et al., 2016; Pečenková et al., 2017). Root hairs increase the contact area of roots with soil and thereby enhance the capacity for nutrient and water uptake. The surface area of the root can be increased many fold by the presence of root hairs (Dittmer, 1937). They are generally short-lived structures and their presence collocates with the maximal absorption of water and nutrients by the root (White, 2012). The length and abundance of root hairs are often correlated with greater uptake and accumulation of mineral elements by roots, especially those with limited mobility in soils, such as inorganic phosphate (Pi), Mn2+, Fe3+, Zn2+ and K+ (Peterson and Steves, 2000; Brown et al., 2013; White, 2013; Salazar-Henao et al., 2016). Phosphate uptake has been studied most intensively, and the calculations of Föhse et al. (1991) suggested that, in soils with low available P, the root hairs might take up 90 % of the P accumulated by a plant. Root hairs are also essential for the formation of the rhizosheath of soil adhering to roots that provides tolerance to a variety of abiotic stresses, including drought and nutrient stresses (Brown et al., 2017). The role of root hairs in the uptake of non-essential elements has been studied less often. However, it has been observed that exposure of arabidopsis to cadmium (Cd) increases root hair density (Bahmani et al., 2016). Cadmium accumulation in soils generally occurs as a result of human activities, including the mining and refining of metal ores and the application of municipal compost and sewage sludge to agricultural soils (White and Greenwood, 2013). Cadmium concentrations <3 μg g–1 dry soil are recommended for agricultural soils to limit Cd concentration in edible produce (White et al., 2012). However, local concentrations close to former mining sites can reach several tens or hundreds of milligrams Cd g–1 dry soil (Alloway, 1995; Banásová et al., 2006, 2008). The availability of mutants with altered root hair initiation or development offers opportunities for studying the effects of root hair abundance on the uptake and accumulation of mineral nutrients and other elements. In the present work, we examined the role of root hairs in Cd uptake by comparing arabidopsis ecotype Columbia (Col-0) with mutants that are unable to develop root hairs (rhd6-1 and cpc/try) or in which most rhizodermal cells develop into hairs (wer/myb23) grown in the presence and absence of Cd. We also examined the development of the endodermis and xylem in these genotypes to determine the effects of other root structures that could affect the uptake and translocation of Cd by plants (Schreiber, 2010; Lux et al., 2011; Barberon, 2017; White and Pongrac, 2017). MATERIALS AND METHODS In vitro cultivation of arabidopsis plants and experimental design Arabidopsis thaliana mutants, which are either completely unable to develop root hairs [rhd6-1 in the ecotype Wassilewskija (Ws)] (Masucci and Schiefelbein, 1994) or form randomly one or two root hairs on the whole primary root surface [cpc/try, from a cross between mutants in the Ws ecotype and the Landsberg erecta (Ler) ecotype; Schellmann et al., 2002], and a double mutant in which most rhizodermal cells develop into hairs (wer/myb23 in the ecotype Col-0) (Kang et al., 2009) were compared with the ecotype Col-0. Schellmann et al. (2002) showed that the trichoblast/atrichoblast ratio was not significantly different in Ws and Ler, and Stetter et al. (2015) found no difference in root hair density between Col-0 and Ws. In the text, rhd6-1 and cpc/try are termed hairless mutants. The ecotype Col-0 has a distinct pattern of alternating files of root hair-forming and non-forming rhizodermal cells, whilst root hair formation is perturbed in the mutants (Fig. 1). Seeds were washed using 0.5 % sodium hypochlorite for 5 min, rewashed three times in sterile distilled water and placed on agar-solidified, hormone-free MS medium (Murashige and Skoog, 1962) containing 1 % sucrose and either 0, 10 or 100 μm Cd applied as Cd(NO3)2. Petri dishes (diameter 90 mm) containing 20 mL of medium were prepared under aseptic conditions. The choice of concentrations was based on preliminary experiments selecting mild 10 μm Cd stress (Cd 10) with limited effect on plant growth and severe stress 100 μm Cd (Cd 100) reducing growth >50 %. In addition we referred to several previous publications with application of these concentrations to arabidopsis and several other species (e.g. Martinez-Peñalver, 2012; Lukačová et al., 2013). The pH of each MS medium was adjusted to 5.8 using HCl or NaOH. A segment of the agar was removed and 15 seeds were sown on the cut surface to avoid contact of shoots with the media. This also enabled the roots to grow into the medium and not on its surface, ensuring homogenous exposure of the rhizodermis and root hairs to the medium. The Petri dishes were oriented vertically and plants were cultivated for 14 days after sowing (DAS). Seeds were stratified 2 d at 4 °C in the dark. Subsequently, Petri dishes were transferred to a controlled-environment growth chamber with a 16/8 h light/dark photoperiod, temperature of 26/20 °C (day/night) and 200 µmol m–2 s–1 light intensity of photosynthetically active radiation (PAR). Five Petri dishes were prepared for each genotype and treatment to provide enough material for all analyses. The experiment was repeated three times. Fig. 1. View largeDownload slide Details of the surface of primary roots (A–D) and sections with root hairs (E–H) of four Arabidopsis thaliana genotypes grown for 14 d in agar containing Murashige and Skoog (MS) medium. The genotype Columbia (Col-0), which has a distinct pattern of alternating root hair-forming and non-forming rhizodermal cells was used as a control (A, E). In the mutant wer/myb23, most epidermal cells develop hairs (B, F), whereas mutants rhd6-1 and cpc/try are unable to form root hairs (C, D and G, H, respectively). Arrows indicate root hairs. Note the development of root hairs in the ‘H position’ facing the intercellular space between two underlying cortical cells in Col-0, whereas the mutant wer/myb23 forms root hairs in both the ‘H position’ and also outside a periclinal cortical cell wall, called the ‘N position’. Scale bars = 80 µm (A–D), 30 µm (E–H). Fig. 1. View largeDownload slide Details of the surface of primary roots (A–D) and sections with root hairs (E–H) of four Arabidopsis thaliana genotypes grown for 14 d in agar containing Murashige and Skoog (MS) medium. The genotype Columbia (Col-0), which has a distinct pattern of alternating root hair-forming and non-forming rhizodermal cells was used as a control (A, E). In the mutant wer/myb23, most epidermal cells develop hairs (B, F), whereas mutants rhd6-1 and cpc/try are unable to form root hairs (C, D and G, H, respectively). Arrows indicate root hairs. Note the development of root hairs in the ‘H position’ facing the intercellular space between two underlying cortical cells in Col-0, whereas the mutant wer/myb23 forms root hairs in both the ‘H position’ and also outside a periclinal cortical cell wall, called the ‘N position’. Scale bars = 80 µm (A–D), 30 µm (E–H). Determination of growth parameters Fourteen days after sowing, plants in the Petri dishes were photographed using a Nikon D90 camera and macro objective AF-S Micro Nikkor 60 mm. Then shoots of ten plants from each Petri dish were carefully excised, washed in distilled water, surface dried with tissue paper and their fresh weight determined. Excised shoots were dried in a VENTI-Line (VWR) drying oven with forced air circulation at a temperature 70 °C for 3 d, and the dry weights of shoots were determined. Primary root length, number of lateral roots and the total length of the root system were evaluated on images of plants using ImageJ software (NIH). Lateral roots were counted and measured if their length was longer than 5 mm. In total, 30 roots from each genotype and treatment were examined. Root hair density and length determination At the end of the experiment, the roots were carefully removed from partially dissolved agar created by increasing the agar temperature temporarily to 40 °C. Roots for anatomical observations were fixed with methanol (Zelko et al., 2012) and stored in a refrigerator for later observations. Root hair length and density vary along the root axis. Therefore, the number of root hairs was counted using a Leica stereomicroscope (Leica M165FC) in three regions of the root: the sub-apical part of the root (2–5 mm from the root tip), a 5 mm long segment in the central part of the root and a 5 mm long segment from the base of the root excluding the hypocotyl junction. The total number of root hairs was calculated as the mean of the three root segments (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated and the data are means of three independent experiments. Cadmium concentration in the shoots The concentration of Cd was determined in shoot tissues using atomic absorption spectrometry (AAS Perkin Elmer Model 1100, at 228.8 nm with deuterium background correction) in the Geoanalytical Laboratories of the Institute of Geomaterials, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. Ten shoots from each Petri dish were combined to get the minimum dry weight for Cd determination. The samples were double step digested in PTFE pressure vessels in a microwave (Anton Paar Multiwave 3000) using concentrated HNO3 and H2O2 at a pressure of 60 bar. Calibration standards were prepared from a stock solution of Cd(NO3)2 (CertiPUR, Merck, Darmstadt, Germany). Tissue Cd concentrations were validated using a certified reference material (CRM NCS DC 73350 Leaves of Poplar, China National Analysis Center for Iron and Steel, Beijing, China). All measurements were performed in triplicate. Analysis of endodermal suberization and xylem lignification Whole-mount samples of shoots stored in methanol were cleared and stained with Fluorol Yellow 088 to identify suberin lamellae (Brundrett et al., 1991; Lux et al., 2005, 2015) and with phloroglucinol-HCl to identify lignification of the xylem. The roots used to identify suberin lamellae were examined under a fluorescence microscope (Axioskop 2 plus, Carl Zeiss, Germany; filter set Carl Zeiss N. 25: excitation filter TBP 400 nm + 495 nm + 570 nm, chromatic beam splitter TFT 410 nm + 505 nm + 585 nm, and emission filter TBP 460 nm + 530 nm + 610 nm) and documented using a digital camera DP72 (Olympus). For measurements, Lucia G 4.80 (LIM, Czech Republic) software was used. The same microscope and camera were used for bright field and/or dark field observation and documentation. In each experiment, at least five primary roots were used for analyses. Statistical analysis Analysis of variance (ANOVA) was performed using Statgraphics Centurion XV.I statistical software. Statistical significance was defined at P <0.05. All data are expressed as the mean ± s.e. for a stated number of replications. All experiments were repeated three times. RESULTS Cadmium accumulation in shoots of wild-type and mutant plants differing in root hair abundance Cadmium concentrations in the shoots of all genotypes increased with increasing Cd concentration in the medium (Fig. 2). Hairy plants (wer/myb23) had greater Cd concentrations in their shoots, and hairless plants (rhd6-1 and cpc/try) had lower Cd concentrations in their shoots, than wild-type plants (Fig. 2). Cadmium concentrations in the shoots of wer/myb23 from the Cd 100 treatment reached 975 μg g–1 d. wt (Fig. 2). Shoots of the hairless mutants rhd6-1 and cpc/try had Cd concentrations of 635 and 636 μg g–1 d. wt, respectively, and shoots of Col-0 had a concentration of 765 μg g–1 d. wt when grown in the Cd 100 treatment. These differences were statistically significant between wer/myb23 and Col-0, and between the hairless mutants and Col-0. In the Cd 10 treatment, wer/myb23 had a shoot Cd concentration of 396 μg g–1 d. wt, whilst the hairless mutants, cpc/try and rhd6-1, had shoot Cd concentrations of 195 and 267 μg g–1 d. wt, respectively. The shoot Cd concentration of wer/myb23 was significantly greater than that of all genotypes, and the shoot Cd concentration of cpc/try was significantly lower than that of Col-0. The differences between Col-0 and rhd6-1 and between rhd6-1 and cpc/try were not significant in the Cd 10 treatment (Fig. 2). Fig. 2. View largeDownload slide Cadmium concentrations, expressed on a dry weight (d. wt) basis, in shoots of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into root hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P < 0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Fig. 2. View largeDownload slide Cadmium concentrations, expressed on a dry weight (d. wt) basis, in shoots of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into root hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P < 0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Growth parameters, root hair length and density Seedlings grown in the absence of Cd developed several leaves during the experiment (Fig. 3). Shoot growth was inhibited by the presence of Cd in the agar, and the magnitude of this inhibition depended on genotype and Cd concentration in the agar (Figs 3 and 4B; Supplementary Data Fig. S1B). Similarly, the length of primary roots was reduced in the presence of Cd, and the magnitude of this effect depended upon the genotype and Cd treatment (Figs 3 and 4A; Supplementary Data Fig. S1A). When plants were grown in the absence of Cd, Col-0 (4.13 cm) and cpc/try (4.29 cm) had the longest primary roots and wer/myb23 (3.61 cm) and rhd6-1 (3.73 cm) had the shortest primary roots. The difference between these two groups was significant (Fig. 4A). The Cd 10 treatment inhibited primary root growth significantly, except in wer/myb23. However, the absolute inhibition of primary root growth in all genotypes was small (Fig. 4A). On the other hand, the Cd 100 treatment resulted in a highly significant reduction in primary root length in all genotypes to approximately one-third of the values observed in the absence of Cd (Fig. 4A). Primary root lengths of Col-0, wer/myb23 and rhd6-1 (1.28, 1.30 and 1.17 cm, respectively) were not significantly different in plants from the Cd 100 treatments. The hairless mutant cpc/try had significantly longer primary roots than other genotypes in both Cd treatments (4.01 cm in Cd 10 and 1.54 cm in Cd 100). There was no positive correlation between primary root length and shoot Cd concentration. Shoot fresh weight was reduced by the presence of Cd in the medium (Fig. 4B). The shoot fresh weight of wer/myb23 was greater than that of other genotypes, whether assayed in the absence or presence of Cd (Fig. 4B). The Cd 10 treatment did not influence shoot fresh weight greatly. However, the Cd 100 treatment reduced shoot fresh weight significantly in all genotypes when compared with the control and the Cd 10 treatment. The reduction in shoot fresh weight was >50 % in all genotypes. Shoot dry weight was also reduced by the presence of Cd in the growth medium (Fig. 4C). Again, wer/myb23 had greater shoot dry weight than other genotypes when assayed under control or Cd 10 conditions (Fig. 4C). Shoot dry weight was least in rhd6-1, whether assayed in the presence or absence of Cd. The effect of the Cd 10 treatment on dry weight was greater than its effect on fresh weight. In the Cd 100 treatment, the shoot dry weight of cpc/try was greater than that of other genotypes. Fig. 3. View largeDownload slide Effects of cadmium on the growth of the ecotype Columbia (Col-0) (A, E, I) the mutant wer/myb23, in which most epidermal cells develop into hairs (B, F, J), and the mutants rhd6-1 (C, G, K) and cpc/try (D, H, L), which are unable to form root hairs. Seeds were placed on the surface of an agar slab placed within Petri dishes oriented vertically to enable shoots to develop without contact with agar and roots to grow within the agar. Plants were cultivated for 14 d in media containing no Cd (Control, C; A–D or either 10 μm Cd (Cd 10; E–H) or 100 μm (Cd 100; I–L) supplied as Cd(NO3)2. Scale bars = 1 cm. Fig. 3. View largeDownload slide Effects of cadmium on the growth of the ecotype Columbia (Col-0) (A, E, I) the mutant wer/myb23, in which most epidermal cells develop into hairs (B, F, J), and the mutants rhd6-1 (C, G, K) and cpc/try (D, H, L), which are unable to form root hairs. Seeds were placed on the surface of an agar slab placed within Petri dishes oriented vertically to enable shoots to develop without contact with agar and roots to grow within the agar. Plants were cultivated for 14 d in media containing no Cd (Control, C; A–D or either 10 μm Cd (Cd 10; E–H) or 100 μm (Cd 100; I–L) supplied as Cd(NO3)2. Scale bars = 1 cm. Fig. 4. View largeDownload slide The effects of cadmium on the (A) primary root length, (B) shoot fresh weight, (C) shoot dry weight, (D) number of lateral roots and (E) total root length of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P <0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Asterisks indicate significant differences between treatments within genotypes. Fig. 4. View largeDownload slide The effects of cadmium on the (A) primary root length, (B) shoot fresh weight, (C) shoot dry weight, (D) number of lateral roots and (E) total root length of Arabidopsis thaliana genotype Columbia (Col-0), a mutant in which most epidermal cells develop into hairs (wer/myb23) and mutants that are unable to form root hairs (rhd6-1 and cpc/try). Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. Three replicate experiments were performed and ten plants were analysed per replicate. Data are expressed as means ± s.e. from three experiments. Statistical significance was defined at P <0.05 determined using analysis of variance (ANOVA). Different letters indicate significant differences between genotypes. Asterisks indicate significant differences between treatments within genotypes. If Cd was taken up preferentially by root cells at the root tip or solely by cells on the surface of the root, then the number of root tips, i.e. the ramification of the root by formation of lateral roots and the total root length of individual genotypes, becomes important (Fig. 4D, E; Supplementary Data Fig. S1D, E). In control conditions, the highest number of laterals (approximately ten per plant) was in Col-0 and cpc/try, followed by rhd6-1 (approximately nine per plant) and wer/myb23 (approximately seven per plant). Even low Cd concentrations in the agar (Cd 10 treatment) reduced the number of lateral roots (Fig. 4D). This occurred in all genotypes, with the greatest decrease (approx. 60 %) being in cpc/try. The Cd 100 treatment had even more of an effect, with an approx. 80 % reduction in the number of lateral roots in Col-0 and a 70 % reduction in wer/myb23 and rhd6-1. In cpc/try, the number of lateral roots was similar in both the Cd 10 and Cd 100 treatments. In the absence of Cd, the total root length, i.e. the sum of the lengths of the primary and all lateral roots of a plant, was greater in rhd6-1 (9.7 cm) and Col-0 (9.3 cm) than in cpc/try (8.4 cm) or wer/myb23 (6.4 cm). The presence of Cd in the agar reduced the total root length of all genotypes. Col-0 had the greatest total root length in the Cd 10 treatment, and cpc/try had the least. In contrast, in the Cd 100 treatment, cpc/try had the greatest total root length and rhd6-1 had the least (Fig. 4E). The hairless mutants rhd6-1 and cpc/try had few root hairs (Fig. 1). In contrast, most rhizodermal cells developed into root hairs in the wer/myb23 mutant, which had about twice as many root hairs per plant as the Col-0 ecotype in all treatments (Table 1). The Cd 10 treatment did not influence the number of root hairs in the wer/myb23 mutant, but reduced the number of root hairs in Col-0 to about 78 % of the number observed in the absence of Cd. The Cd 100 treatment reduced the number of root hairs to about 34 % of their number in the absence of Cd in both wer/myb23 and Col-0 (Table 1). The number of root hairs per millimetre increased from 30 in the absence of Cd to 43 in the Cd 100 treatment in wer/myb23 and from nine in the absence of Cd to 20 in the Cd 100 treatment in Col-0. The presence of Cd, especially the 100 Cd treatment, induced the frequent formation of irregularly occurring, unusually shaped, balloon-like, bulbous or pear-like root hairs in both wer/myb23 and Col-0 (Fig. 5A, B). In the hairless cpc/try mutant, occasional swelling of rhizodermal cells occurred (Fig. 5C). Fig. 5. View largeDownload slide The effect of 100 μm Cd(NO3)2 on the morphology of the rhizodermis and root hairs of (A) the ecotype Col-0, (B) the wer/myb23 mutant and (C) the cpc/try mutant. Abnormally shaped, balloon-like root hairs (arrows) occurred in both Col-0 and wer/myb23. Swollen rhizodermal cells (arrowheads) occurred occasionally in cpc/try. Scale bars = 100 μm. Fig. 5. View largeDownload slide The effect of 100 μm Cd(NO3)2 on the morphology of the rhizodermis and root hairs of (A) the ecotype Col-0, (B) the wer/myb23 mutant and (C) the cpc/try mutant. Abnormally shaped, balloon-like root hairs (arrows) occurred in both Col-0 and wer/myb23. Swollen rhizodermal cells (arrowheads) occurred occasionally in cpc/try. Scale bars = 100 μm. Table 1. The effects of cadmium on the number of root hairs per plant of Arabidopsis thaliana ecotype Columbia (Col-0) and a mutant in which most epidermal cells develop into hairs (wer/myb23) Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. The total number of root hairs was calculated as the mean of the three root segments viewed (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated. Data are expressed as means ± s.e. from three independent experiments. View Large Table 1. The effects of cadmium on the number of root hairs per plant of Arabidopsis thaliana ecotype Columbia (Col-0) and a mutant in which most epidermal cells develop into hairs (wer/myb23) Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Control Cd 10 Cd 100 Col-0 4637 ± 103 3652 ± 76 1580 ± 47 wer/myb23 8327 ± 210 8279 ± 288 2827 ± 97 Plants were cultivated for 14 d in agar containing either no Cd (control), 10 μm (Cd 10) or 100 μm (Cd 100). Cadmium was supplied as Cd(NO3)2. The total number of root hairs was calculated as the mean of the three root segments viewed (sub-apical, central and basal) multiplied by the total root length. A minimum of five roots from each genotype and treatment were evaluated. Data are expressed as means ± s.e. from three independent experiments. View Large Anatomical characteristics that might influence Cd uptake and translocation It has been proposed that Cd might reach the xylem, and thence the shoot, by either an apoplasmic or a symplasmic pathway across the root (Lux et al., 2011). The barrier to apoplasmic transport of solutes to the stele is at the endodermis (White, 2001; Lux et al., 2011; Barberon, 2017). The first stage in the development of the endodermis is the formation of Casparian bands, which is followed closely by the second stage in which suberin lamellae cover the whole inner surface of endodermal cells. The formation of the Casparian band restricts transport through the apoplast to the stele, and the suberization of endodermal cells prevents solute transport across the plasma membrane of endodermal cells, thereby ensuring symplasmic transport to the stele (White, 2001; Moore et al., 2002; Lux et al., 2011; Barberon, 2017). The rate of development of the endodermis, and its response to Cd in the rhizosphere, differs between Col-0, wer/myb23, rhd6-1 and cpc/try (Fig. 6). In the absence of Cd in the rhizosphere the distance of suberin lamellae from the root tip in Col-0 was similar to that in the hairless mutants rhd6-1 and cpc/try. Suberin deposition began about 2.8 mm from the root tip, and well-developed suberin lamellae were formed at about 3.3 mm from the root tip. In wer/myb23, these distances were about 1 mm further from the root tip (Figs 6 and 7). Development of the endodermis differed little between the control and the Cd 10 treatment, although suberization of the endodermis in wer/myb23 occurred closer to the root tip in the Cd 10 treatment than in the absence of Cd. The Cd 100 treatment resulted in the development of the endodermis closer to the root tip in all genotypes. In Col-0 and wer/myb23, suberization of the endodermal cells occurred 1–1.5 and 1.5–2 mm from the tip, respectively. In the hairless mutants, rhd6-1 and cpc/try, suberization of the endodermal cells occurred further away from the root tip, at 2.2–2.8 mm from the root tip in rhd6-1 and at 2.8–3.3 mm from the root tip in cpc/try (Figs 6 and 7). In these hairless mutants, irregular and patchy formation of suberin lamellae took place along an extended length of the root, which did not occur in Col-0 or wer/myb23. Fig. 6. View largeDownload slide Graphical summary of the effects of increasing agar Cd concentration on suberization of the endodermis and xylem development in roots of the Arabidopsis thaliana ecotype Columbia (Col-0), wer/myb23, a mutant in which most epidermal cells develop into hairs, and the mutants rhd6-1 and cpc/try that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C) and either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. The root anatomy of each genotype is shown in cross-section at the top of the figure according to the basic scheme of Salazar-Henao et al. (2016). The changing position of root hair initiation, onset of protoxylem differentiation (red line) and deposition of suberin lamellae (the beginning of suberin lamellae deposition is indicated by a dashed green line and complete suberin lamellae deposition by a solid green line) in the endodermis at the root apices of the four genotypes are shown in longitudinal views. Fig. 6. View largeDownload slide Graphical summary of the effects of increasing agar Cd concentration on suberization of the endodermis and xylem development in roots of the Arabidopsis thaliana ecotype Columbia (Col-0), wer/myb23, a mutant in which most epidermal cells develop into hairs, and the mutants rhd6-1 and cpc/try that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C) and either 10 μm (Cd 10) or 100 μm (Cd 100) supplied as Cd(NO3)2. The root anatomy of each genotype is shown in cross-section at the top of the figure according to the basic scheme of Salazar-Henao et al. (2016). The changing position of root hair initiation, onset of protoxylem differentiation (red line) and deposition of suberin lamellae (the beginning of suberin lamellae deposition is indicated by a dashed green line and complete suberin lamellae deposition by a solid green line) in the endodermis at the root apices of the four genotypes are shown in longitudinal views. Fig. 7. View largeDownload slide Effects of increasing Cd concentration on the formation of root hairs and the development of suberin lamellae in the endodermis of the Arabidopsis thaliana ecotype Columbia (Col-0), (A, B, I, J); wer/myb23, a mutant in which most epidermal cells develop into hairs (C, D, K, L); and the mutants rhd6-1 (E, F, M, N) and cpc/try (G, H, O, P) that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C), (A, C, E, G) or 100 μm (Cd 100), (B, D, F, H) supplied as Cd(NO3)2. Arrows indicate the beginning of suberin lamellae deposition in the endodermis. (I–P) Detail of the root in which suberization of the endodermis has just begun. Note the patchy development of suberin lamellae (occurring in individual endodermal cells) in mutant rhd6-1 (N). Scale bars = 600 μm (A–H), 80 μm (I–P). Fig. 7. View largeDownload slide Effects of increasing Cd concentration on the formation of root hairs and the development of suberin lamellae in the endodermis of the Arabidopsis thaliana ecotype Columbia (Col-0), (A, B, I, J); wer/myb23, a mutant in which most epidermal cells develop into hairs (C, D, K, L); and the mutants rhd6-1 (E, F, M, N) and cpc/try (G, H, O, P) that are unable to form root hairs. Plants were cultivated for 14 d in agar containing no Cd (C), (A, C, E, G) or 100 μm (Cd 100), (B, D, F, H) supplied as Cd(NO3)2. Arrows indicate the beginning of suberin lamellae deposition in the endodermis. (I–P) Detail of the root in which suberization of the endodermis has just begun. Note the patchy development of suberin lamellae (occurring in individual endodermal cells) in mutant rhd6-1 (N). Scale bars = 600 μm (A–H), 80 μm (I–P). Development of xylem, with lignified cell walls of protoxylem developing in the periphery of the central cylinder, generally coincides with root hair development (Demchenko and Kalimova, 2008). In the hairless mutants rhd6-1 and cpc/try, development of the xylem started closer to the root tip than in Col-0 and the wer/myb23 mutant in the Cd 10 treatment. DISCUSSION It is the aim of many crop scientists to supply sufficient, nutritious food and fodder for the future (White et al., 2012). It is also essential that both food and fodder are safe to eat. Cadmium is toxic to all living organisms, and its presence, accumulation and effect on plants has been intensively studied. Research is focused on the development of crops that can tolerate Cd in the environment and accumulate less in their edible parts or to develop plants that can accumulate Cd for the phytoremediation of contaminated land (Prasad, 1995; Das et al., 1997; McGrath et al., 2001; Lux et al., 2004, 2011; Van Belleghem et al., 2007; Lukačová-Kuliková and Lux, 2010; Singh et al., 2011; Vaculík et al., 2012; White et al., 2012; Martinka et al., 2014; White and Pongrac, 2017). The present study shows that shoots of the hairy mutant, wer/myb23, had greater Cd concentrations than shoots of the ecotype Col-0 and that shoots of both wer/myb23 and Col-0 had greater Cd concentrations than shoots of the hairless mutants rhd6-1 and cpc/try (Fig. 2). The shoot Cd concentrations reported here are relatively high compared with previous studies of arabidopsis and other species exposed to Cd (Bovet et al., 2003; Lukačová-Kuliková and Lux, 2010). One reason for this might be that the availability of Cd is greater from agar than from soil substrates. All genotypes showed inhibition of root growth and reduced shoot biomass as a result of Cd treatment (Figs 3 and 4), which is consistent with the toxicity of Cd to plants (Prasad, 1995; Lux et al., 2011; White and Pongrac, 2017). However, genotypes differed in their root development and shoot biomass both in the absence and in the presence of Cd (Figs 3 and 4). The hairy mutant, wer/myb23, had a shorter primary root, formed fewer lateral roots and had a shorter total root length than Col-0 in the absence of Cd and in the Cd 10 treatment (Fig. 4A, D, E), but had a greater shoot fresh weight and dry weight under these conditions, as well as in the Cd 100 treatment (Fig. 4B, C). The hairless mutant, rhd6-1, although in ecotype Ws, behaved similarly to Col-0 in both root development and shoot biomass, whereas the hairless mutant cpc/try developed longer primary roots, more lateral roots and a greater total root length than other genotypes in the Cd 100 treatment. The hairless double mutant cpc/try had greater shoot fresh weight than Col-0 in the Cd 10 treatment and more shoot dry weight than Col-0 in the Cd 100 treatment. The total number of root hairs was about twice as great in the hairy mutant wer/myb23 than in Col-0 (Table 1). In both wer/myb23 and Col-0, root hairs developed closer to the root apex in the Cd 100 treatment (Fig. 6). Differences in the shoot phenotypes of the two hairless genotypes might occur because of their different genetic backgrounds, rhd6-1 being derived from ecotype Ws and cpc/try from a cross between mutants in Ws and Ler ecotypes. However, the data reveal no differences between the hairless genotypes in their shoot Cd concentrations (Fig. 2). Recent studies have indicated a significant contribution of root hairs to the uptake of some toxic elements. Zheng et al. (2011) concluded that root hairs contributed significantly to Cd uptake by barley, and Balestri et al. (2014) noted a possible involvement of root hairs in Cd uptake by Pteris vittata. In arabidopsis, the number and length of root hairs in the apical 5 mm of the root increased in the presence of Cd (Bahmani et al., 2016). This is consistent with the results obtained here for both Col-0 and wer/myb23. However, it should be noted that, due to the reduced total root length, the total number of root hairs per plant was reduced (Table 1). In the Cd 10 treatment, the Cd content of the shoot was strongly correlated with the number of root hairs per plant across all genotypes studied (Fig. 7), suggesting a contribution of root hairs to Cd uptake and the presence of a symplasmic pathway to the xylem under these conditions. However, the lack of a correlation between the Cd content of the shoot and the number of root hairs in the Cd 100 treatment suggests that both symplasmic and apoplasmic pathways contribute to Cd delivery to the xylem when the rhizosphere Cd concentration is large (Fig. 7). When arabidopsis roots are exposed to Cd, it accumulates in both the apoplasmic and symplasmic space (van Beleghem et al., 2007). Several studies have indicated the importance of the development of apoplasmic barriers in the endodermis and exodermis in restricting Cd uptake by roots and Cd accumulation in the shoot (Lux et al., 2004, 2011; Ďurčeková et al., 2007; Vaculík et al., 2009; Lukačová-Kuliková and Lux, 2010). Ontogenesis of both these highly specialized root layers is characterized by three stages: (1) the formation of Casparian bands close to the root tip; (2) the development of suberin lamellae around endodermal cells; and (3) the thickening of cell walls of endodermal cells (von Guttenberg, 1968; White, 2001; Schreiber, 2010; Barberon, 2017). Variation in the development of the endodermis has been observed among ecotypes of wild species and cultivars of crops, and correlated with Cd accumulation in the shoot (Lux et al., 2004; Lukačová-Kuliková and Lux, 2010; Vaculík et al., 2012; Tao et al., 2017). The translocation of Cd to the shoot is lower in genotypes with accelerated development of apoplasmic barriers, close to the root tip, when compared with genotypes developing these barriers more distant from the root tip. The development of the endodermis is extremely sensitive to environmental conditions (Redjala et al., 2011; Lux et al., 2011; Líška et al., 2016), and several studies have shown that abiotic stresses, including the presence of Cd in the rhizosphere, induce early formation of apoplasmic barriers by premature deposition of suberin and lignin to the cell walls (Reinhardt and Rost, 1995; Schreiber et al., 1999; Enstone et al., 2002; Lux et al., 2011; Balestri et al., 2014; Martinka et al., 2014; Líška et al., 2016). In a previous study, unilateral exposure of maize roots to Cd resulted in earlier development of apoplasmic barriers in the side of the root exposed to Cd (Lux et al., 2011). In the present experiments, it was observed that exposure of roots to the Cd 100 treatment resulted in suberin lamellae being formed closer to the root tip (Fig. 6). It is possible that this might restrict the uptake of Cd and Cd accumulation in shoots. This is consistent with the lower Cd content of shoots of Col-0 than of shoots of rhd6-1 and cps/try in the Cd 100 treatment (Fig. 7). However, it is inconsistent with the greater Cd content of shoots of wer/myb23 than shoots of rhd6-1 and cps/try in the Cd 100 treatment (Fig. 7). Thus, in addition to Cd movement to the xylem via an apoplasmic pathway, the involvement of a symplasmic pathway to the xylem facilitated by root hairs might be suggested even in the Cd 100 treatment. The irregular and patchy development of suberin lamellae in rhd6-1 and also the very early development of protoxylem close to the root tip in this genotype might also contribute to the differences in Cd accumulation in the shoot among the genotypes studied. Shorter hairs are formed in roots inside agar compared with longer and thin hairs of roots growing on the agar surface, developing without contact with Cd-contaminated agar only in the humid air (Fig. 8). Additionally, abnormal development of root hairs was observed in both Col-0 and wer/myb23 in the Cd 100 treatment, suggesting a direct effect of Cd on hair initiation and tip growth (Fig. 5). These processes are regulated by plant hormones, Ca channels at the apex of the hair and cytoskeletal rearrangements (Baluška et al., 2000; Ryan et al., 2001; Scheifelbein et al., 2014; White, 2015), and are sensitive to various environmental stresses (Mueller and Schmidt, 2004; Balcerowicz et al., 2015; Bahmani et al., 2016). Cell wall visco-elastic properties can also be affected by toxic elements (Ma et al., 2004; Krupinski et al., 2016). Fig. 8. View largeDownload slide Difference in development of root hairs (arrows) of Arabidopsis thaliana ecotype Columbia (Col-0) cultivated in vitro with roots growing (A) within agar-solidified MS medium (agar) or (B) on the surface of the same medium (air). Note the difference in the root hair length and thickness. Scale bars = 70 μm. Fig. 8. View largeDownload slide Difference in development of root hairs (arrows) of Arabidopsis thaliana ecotype Columbia (Col-0) cultivated in vitro with roots growing (A) within agar-solidified MS medium (agar) or (B) on the surface of the same medium (air). Note the difference in the root hair length and thickness. Scale bars = 70 μm. CONCLUSIONS Shoots of the hairy genotype, wer/myb23, had greater Cd concentrations, and shoots of the hairless mutants, rhd6-1 and cpc/try, had lower Cd concentrations, than shoots of the ecotype Col-0 in both Cd 10 and Cd 100 treatments. In the Cd 10 treatment, the Cd content of the shoot was strongly correlated with the number of root hairs per plant across all genotypes studied, suggesting a contribution of root hairs to Cd uptake and that a symplasmic pathway contributed to Cd movement to the xylem under these conditions. The Cd content of the shoot was weakly correlated with the number of root hairs in the Cd 100 treatment, suggesting that the symplasmic pathway contributed less to the movement of Cd to the xylem at greater rhizosphere Cd concentrations. It is suggested that the development of the endodermis closer to the root apex in Col-0 and wer/myb23 reduces the potential apoplasmic movement of Cd to the xylem in these genotypes relative to rhd6-1 and cpc/try, which form their endodermis at a greater distance from the root apex. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Figure S1: the effects of cadmium on the primary root length, shoot fresh weight, shoot dry weight, number of lateral roots and total root length of Arabidopsis thaliana genotypes Col-0, wer/myb23, rhd6-1 and cpc/try. Figure S2: correlation between the Cd content of the shoot and number of root hairs per plant. ACKNOWLEDGEMENTS This work was supported by the Slovak Research and Development Agency, contract No. APVV SK-AT-2015-0009, by the Slovak Grant Agency VEGA, grant no. VEGA 1/0605/17, the Austrian Science Fund ANR-FWF I 1725-B16 and the Scientific & Technological Cooperation (WTZ) Austria & Slovakia SK 04/2016. We thank Kai Dünser for providing rhd6-1, cpc/try and wer/myb23 seeds. The reviewers are thanked for their helpful comments. P.J.W. was supported by the Rural & Environment Science & Analytical Services Division of the Scottish Government. This paper as a part of Special Issue is dedicated to Peter Barlow, our dear colleague and friend. LITERATURE CITED Alloway BL . 1995 . Heavy metals in moils . 2nd edn . Glasgow, UK : Blackie Academic and Professional an imprint of Chapman and Hall . Google Scholar Crossref Search ADS Bahmani R , Kim DG , Kim JA , Hwang S . 2016 . 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Annals of Botany – Oxford University Press

Published: Jan 31, 2018

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