*3.2. Effects of Pb on Water-Grown Seedlings*

Rice is a semiaquatic plant and its coleoptile grows faster underwater than in air [35,36]. In contrast, the root formation and the growth of the primary leaf inside the coleoptile are strongly suppressed when caryopses are germinated underwater [35]. As shown in Figure 4A, the root formation was strongly suppressed under submerged conditions, even in the absence of Pb. When germinated caryopses were grown for 4 days underwater, the length of the control coleoptiles reached approximately 35 mm (Figure 4B). In contrast to the air-grown seedlings, the growth of water-grown coleoptiles was significantly inhibited by the treatment with 0.25 mM Pb and the inhibitory effect increased with increasing Pb concentration. Coleoptile growth was reduced by about 50% by the 0.5 mM Pb treatment (Figure 4B).

The effects of Pb treatment at 0.5 mM on cell wall extensibility and the cellular osmotic concentration of water-grown coleoptiles are shown in Figure 4C,D. The Pb treatment significantly decreased cell wall extensibility, but did not affect the cellular osmotic concentration. Furthermore, when seedlings grown under submerged conditions for 2 days without Pb were transferred to the Pb-containing medium, Pb at 0.5 mM significantly inhibited the coleoptile growth afterward and it also significantly lowered cell wall extensibility (Table 1).

**Figure 4.** Effects of Pb on the growth of coleoptiles (**A**,**B**), the cell wall extensibility (**C**), and the cellular osmotic concentration (**D**) in coleoptiles of water-grown rice seedlings. Germinated caryopses were submerged in a 2 mM MES-KOH buffer (pH 6.0) with or without different concentrations of Pb and then grown for 4 days in the dark. (**A**) Photograph showing seedlings grown for 4 days under submerged conditions. Scale bar = 1 cm. (**B**) The length of water-grown coleoptiles. Data are means ± SE (*n* = 18). Different letters above the bars represent statistically significant differences (Tukey's HSD test, *p* < 0.05). (**C**) The cell wall extensibility of the upper region of coleoptiles grown for 4 days with or without 0.5 mM Pb was measured. Data are means ± SE (*n* = 16–18). \* Mean values were significantly different between the control and Pb treatment (Student's *t*-test, *p* < 0.05). (**D**) The osmotic concentration of the cell sap obtained from coleoptiles that had been grown for 4 days with or without 0.5 mM Pb was measured. Data are means ± SE (*n* = 3).

**Table 1.** Effects of Pb on the growth and cell wall extensibility of water-grown rice coleoptiles. Germinated caryopses were grown for 2 days in a 2 mM MES-KOH buffer (pH 6.0) (Initial), and then the seedlings were immediately transferred to the same buffer with or without 0.5 mM Pb. The transferred seedlings were grown for a further 2 days (the control and Pb treatment). The cell wall extensibility of the upper region of the coleoptile was measured. Data are means ± SE (*n* = 15–18). \* Mean values were significantly different between the control and Pb treatment (Student's *t*-test, *p* < 0.05).


The amount of cell wall polysaccharides and cell wall-bound phenolic acids in watergrown coleoptiles (Figure 5A,B) were smaller than those in air-grown ones (Figure 3A,B). On the basis of unit length and unit fresh weight of the coleoptile, Pb treatment at 0.5 mM significantly increased the amounts of matrix polysaccharides and cellulose (Figure 5A). The amounts per unit matrix polysaccharide content of phenolic acid monomers and dimers were lower in Pb-treated coleoptiles than in control ones, particularly the amounts of *p*-CA and 8-*O*-4 DFA in Pb-treated coleoptiles, which were significantly lower than those in control ones (Figure 5B). The effects of Pb on the amounts of cell wall constituents were similar in air- and water-grown coleoptiles.

**Figure 5.** Effects of Pb on the amounts of cell wall polysaccharides (**A**) and cell wall-bound phenolic acids (**B**) in water-grown rice coleoptiles. Coleoptiles were grown under submerged conditions for 4 days in the presence or absence of 0.5 mM Pb. (**A**) The sugar content in each cell wall fraction was determined by the phenol-sulfuric acid method. Amounts of cell wall polysaccharides were expressed on the basis of unit length and unit fresh weight (FW) of the coleoptile. MP, matrix polysaccharides; CL, cellulose. (**B**) Phenolic acids were analyzed by the HPLC and their amounts were expressed on the basis of unit matrix polysaccharide (MP) content. *p*-CA, *p*-coumaric acid; FA, ferulic acid; DFAs, diferulic acids. Data are means ± SE (*n* = 3). \* Mean values were significantly different between the control and Pb treatment (Student's *t*-test, *p* < 0.05).

#### **4. Discussion**

Plant cell expansion is caused by the influx of water into the cell and the osmotic concentration of the cell sap provides the driving force for water uptake. In this context, an increase in osmotic concentration of the cell sap is expected to promote the growth rate, while a decrease slows it down. The cellular osmotic concentration, along with cell wall extensibility, is thought to be involved in the regulation of plant growth. In the present

results, Pb had no negative effect on the osmotic concentration in air- and water-grown coleoptiles (Figures 2B and 4D), suggesting that the cellular osmotic concentration is not related to the growth inhibition induced by Pb. In contrast, the Pb treatment decreased cell wall extensibility of both air- and water-grown coleoptiles, when it inhibited their growth (Figures 1, 2A and 4B,C, Table 1). It is suggested by these results that the decrease in the ability of the cell wall to extend is associated with the inhibition of coleoptile growth in response to the Pb exposure. The relationship between the growth inhibition of stem organs and the decrease in cell wall extensibility has been extensively examined in studies of environmental stimuli and plant hormones on stem growth [17,30,37–39].

The quantitative changes in cell wall constituents underlie the modification of mechanical properties of the cell wall [15,17]. The amounts of cell wall polysaccharides per unit fresh weight and per unit length of the stem show the proportion and the cross-sectional mass of the cell wall in the stem organ, respectively, and thus those values are thought to represent the thickness of the cell wall [28]. On these bases, the amounts of matrix polysaccharides and cellulose in Pb-treated coleoptiles were higher than those in control ones in both cultivation conditions (Figures 3A and 5A), indicating that the cell wall thickness of Pb-treated coleoptiles was greater than that of control ones. The increase in the thickness of the cell walls results in a decrease in the cell wall extensibility of stem organs [28,30,37,40]. Therefore, Pb-induced cell wall thickening may be primarily involved in the decrease in cell wall extensibility in rice coleoptiles.

In addition to cell wall polysaccharides, the increases in the amounts of DFA and FA were associated with a decrease in the ability of the cell wall to extend in gramineous shoots [20,34,41,42]. The present results, however, showed that the levels of cell wallbound DFAs, FA, and *p*-CA in Pb-treated coleoptiles were lower than those in control ones (Figures 3B and 5B), suggesting that Pb decreased the concentration of DFA-mediated cross-linkages within cell wall architecture. These results suggest that cell wall-bound phenolic acids were not involved in the Pb-mediated decrease in cell wall extensibility. As for the effect of Pb on cell wall-bound phenolic acids, the Pb treatment only slightly affected the ratio of the total amount of three DFA isomers to the amount of FA, which was 0.20 and 0.19 for the control and Pb-treated coleoptiles grown in air and 0.15 and 0.14 for the control and Pb-treated ones grown underwater, respectively (calculated using data in Figures 3B and 5B). These results suggest that Pb scarcely affects the coupling step of FA to produce DFA. Therefore, the decreases in DFA levels in Pb-treated coleoptiles may be attributed to the reduced FA level. The Pb treatment decreased the amounts of both FA and *p*-CA that are synthesized via the phenylpropanoid pathway [43]. Therefore, Pb may affect the reactions in the pathway and/or the feruoylation and coumaroylation of matrix polysaccharides, such as arabinoxylans [20,44,45].

Plant cell walls are able to bind metal cations and a large number of heavy metals incorporated into plants were localized in the cell walls [2,46,47]. Therefore, plant cell walls function not only as a barrier limiting the penetration of heavy metals but also as a sink for the accumulation of heavy metals [23]. Plant cell walls serve to sequester heavy metals from the cytoplasm, as do phytochelatins and metallothioneins, proteins that bind heavy metals. Because the cell wall can accumulate and immobilize a significant amount of heavy metals, Pb-induced cell wall thickening is thought to enhance the defense mechanism against the Pb stress [23]. The present study showed that Pb induced the cell wall thickening in coleoptiles and that the thickening caused a decrease in cell wall extensibility. Therefore, Pb-induced cell wall thickening may contribute not only to the defense strategy against Pb stress but also to the growth regulation of the aboveground organ by modifying the cell wall's mechanical properties.

At present, the mechanism by which Pb promotes cell wall thickening in coleoptiles is not clarified. Pb treatment stimulated the production of ROS in plant cells [7,8,48]. Although ROS have toxic effects on cellular functions, they act as signaling molecules in stress-induced cellular responses in plants. Among ROS, hydrogen peroxide is thought to be involved in the structural modification processes of the cell wall in response to abiotic

stresses [49]. The application of hydrogen peroxide affected plant growth responses, such as root gravitropism [50]. Therefore, it is conceivable that hydrogen peroxide signaling may be involved in Pb-induced cell wall thickening in rice coleoptiles. In addition, Pb may affect the autolytic activity of the cell wall. Plant cell walls contain various kinds of enzymes that are involved in cell wall remodeling [51,52]. Since Pb is able to bind to acidic sugar residues of matrix polysaccharides [23,46], it is likely that Pb accumulated within the cell walls may interfere with the action and activity of enzymes involved in the degradation of cell wall polysaccharides. This possibility remains to be clarified in a future study.

Pb strongly inhibits root growth. So, it has been believed that the inhibitory effect of Pb on the growth of plant aboveground organs is attributed to the inhibition of root development [2]. The present results showed that Pb inhibited the coleoptile growth of water-grown seedlings, which did not develop roots (Figure 4A), suggesting that Pb directly inhibits the growth of aboveground organs. In the case of rice caryopses, the cell division ceases about 60 h after sowing and the coleoptile growth afterward is mainly due to cell elongation [36]. Therefore, the results of the transplant experiment (Table 1) suggest that Pb inhibited the cell elongation process of coleoptiles by reducing cell wall extensibility. In air-grown seedlings, the effective concentration of Pb for the inhibition of coleoptile growth was much higher than that of root growth (Figure 1). However, the dose–response of Pb for the inhibition of the growth of water-grown coleoptiles was similar to that of air-grown roots (Figure 6). Furthermore, when concentrations of Pb in the cytoplasmic fluid were elevated to the order of ten μM, severe growth inhibition was observed in shoots and roots of air-grown seedlings. It is suggested by these results that there are no apparent differences in the organ susceptibility to Pb between aboveground organs and roots in rice seedlings.

**Figure 6.** Dose–response curves of Pb on the growth of coleoptiles of air- and water-grown seedlings and of roots of air-grown seedlings. Lengths of coleoptiles and roots are shown as a percentage of the control (0 mM Pb) value. Values were calculated using the data in Figures 1 and 4B. Mean values are shown.

#### **5. Conclusions**

Pb treatment increases the thickness of the cell walls of rice coleoptiles irrespective of cultivation conditions, which may decrease cell wall extensibility. The decrease in cell wall extensibility is associated with the inhibition of coleoptile growth. It is conceivable that the growth inhibition of stem organs by other heavy metals also involves cell wall remodeling similar to that of Pb. Finally, water-grown rice seedlings may serve as a good experimental system to investigate how the cell walls in the aboveground organs of plants resist the penetration of heavy metals into the cells.

**Author Contributions:** K.W., K.S. and T.H. designed the research; K.W., K.S. and H.M. performed experiments; K.W., K.S., T.H. and H.M. analyzed data; K.W. and T.H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Acknowledgments:** We thank Kazuya Izumoto, Yui Kagotani, Masashi Ueda, and Saki Terazawa of our laboratory for their efforts and cooperation during the course of the present study. We also thank Tomomi Shimonaka of Osaka Metropolitan University for excellent technical assistance in ICP-MS analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
