**1. Introduction**

Lead (Pb) is one of the most abundant heavy metal contaminants in both terrestrial and aquatic environments. Pb is not biodegradable and accumulates in organisms. Pb has no biological function, but it is highly toxic to living organisms even at low concentrations and causes disease, such as neurotoxicity and kidney damage in animals [1]. In plants, a prominent Pb toxicity is the inhibition of organ growth [2–5]. The toxic effects of Pb on cellular functions have been extensively studied; for example, incorporated Pb stimulates the production of reactive oxygen species (ROS), elevates the levels of lipid hydroperoxide, and increases the activities of antioxidant enzymes, while it decreases the chlorophyll contents and thus declines the photosynthetic activity [2–10]. Alterations in metabolic and biochemical processes may cause physiological changes in plant development under Pb stress conditions. In addition to metabolic and biochemical alterations, it has been shown that Pb disturbed the microtubule organization in meristem cells and interfered with cell

**Citation:** Wakabayashi, K.; Soga, K.; Hoson, T.; Masuda, H. The Modification of Cell Wall Properties Is Involved in the Growth Inhibition of Rice Coleoptiles Induced by Lead Stress. *Life* **2023**, *13*, 471. https:// doi.org/10.3390/life13020471

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 27 December 2022 Revised: 6 February 2023 Accepted: 6 February 2023 Published: 8 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

division in roots [11,12]. Therefore, the suppression of cell division in root meristem may be associated with the inhibition of root growth under the Pb stress condition. In addition to the increment of cell number, the increase in cell volume is an important factor determining the growth rate of plant organs. Lane et al. [13] showed that Pb interfered with auxininduced cell elongation in segments of wheat (*Triticum aestivum*) coleoptiles. They also showed that Pb treatment decreased the deformation ability of turgid coleoptile segments under constant inflection load. These results imply that Pb affected the mechanical properties of the cell walls when it inhibited auxin-induced growth of the segments. However, it has not been clarified whether Pb directly affects the cell wall mechanical properties in growing stem organs.

Plant cell walls surround each protoplast and provide protoplasts with mechanical rigidity. Furthermore, cell walls play an important role in the regulation of growth and morphogenesis in plants [14–16]. Cell wall extensibility, a parameter of cell wall mechanical property, represents the capacity of the cell wall to extend and thus the parameter is related to the elongation capacity of plant cells [15–17]. Cell walls of growing plant tissues are mainly composed of polysaccharides, such as cellulose and a variety of matrix polysaccharides. The quantities and chemical structures of cell wall polysaccharides are considered to be factors determining cell wall extensibility [14,15,17,18]. In addition to polysaccharides, the cell wall of gramineous (cereal) plants, such as rice (*Oryza sativa*), wheat, and maize (*Zea mays*), contain a significant amount of phenolic acid monomers, such as ferulic acid (FA), which are ester-bound to matrix polysaccharides [19,20]. Some FA residues undergo a coupling reaction to produce diferulic acid (DFA), which forms cross-links between matrix polysaccharides [21,22]. The formation of the cross-linkages by phenolic acids makes the cell wall mechanically rigid [20].

Plant cell walls play an efficient barrier to the entry of heavy metals into the protoplast [23]. Heavy metals, including Pb, increased the thickness of root cell walls in several plant species, such as *Vicia faba*, *Oryza sativa*, and *Allium cepa*, and protonemata cells of *Funaria hygrometrica* [24–27]. The thickening of the cell wall is associated with a decrease in cell wall extensibility [15,17,28]. An increase in cell wall thickness in seedling stems is accompanied by an increase in cell wall constituents, especially polysaccharides [17,28]. It is expected that Pb increases levels of cell wall constituents, such as polysaccharides and cell wall-bound DFA and FA in coleoptiles, which may promote the cell wall thickening and the formation of cross-linkages within the cell wall and thereby decrease cell wall extensibility. In the present study, we investigated the above possibility using air- and water-grown rice coleoptiles. The present results revealed a key role of the plant cell wall in the regulation of organ growth under heavy metal stress conditions.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Growth Conditions*

Caryopses of rice (*Oryza sativa* L. cv. Koshihikari) were sterilized in ca. 1% (*v*/*v*) sodium hypochlorite solution for 1 h and then soaked in deionized water for two days at 25 ◦C in the dark. Germinated caryopses were grown for four days in the dark at 25 ◦C under two different cultural conditions: on moistened filter paper (air-grown) and underwater (water-grown). For the cultivation in air, germinated caryopses were placed on one layer of filter paper in a cylindrical polycarbonate box (15 cm in diameter and 8 cm in height) which contained 30 mL of 2 mM MES-KOH buffer (pH 6.0) containing different concentrations (0, 0.1, 0.3, 1.0, and 3.0 mM) of PbCl2 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cultivation underwater was as follows: germinated caryopses were submerged in a test solution (ca. 7 cm in depth) in a polycarbonate cylinder (4 cm in diameter and 11 cm in height). Each cylinder contained 80 mL of 2 mM MES-KOH buffer (pH 6.0) containing different concentrations (0, 0.25, 0.5, and 1.0 mM) of PbCl2. For the transplant experiment, water-grown seedlings that had been grown for 2 days in the dark at 25 ◦C in 2 mM MES-KOH buffer (pH 6.0) were immediately transferred to the same buffer containing 0 and 0.5 mM PbCl2 and grown for a further 2 days in the

same conditions. On the days after planting, the lengths of coleoptiles and roots were measured with a commercially obtainable ruler. After the measurement, coleoptiles were excised. Since the coleoptile excised from air-grown seedlings contained a primary leaf inside, a vertical slit was made at the basal portion of the coleoptile and then the primary leaf was removed using forceps. After the removal of the primary leaf, the fresh weight of the coleoptile was measured using an electric balance. In contrast, the primary leaf inside the coleoptile scarcely grew under submerged conditions. So, the coleoptile excised from water-grown seedlings was used readily for the measurement of fresh weight. All manipulations were performed under dim green light (ca. 0.09 μmol m−<sup>2</sup> s−<sup>1</sup> at handling level). The growth experiment was repeated at least three times. The amounts of cell wall constituents, the cellular osmotic concentration, and the Pb content were determined using three samples obtained from three independent experiments. The measurement of the cell wall mechanical properties was repeated twice using samples obtained from two independent experiments.

### *2.2. Assay of Pb Content*

Shoots consisted of coleoptile and the inner primary leaf and roots were used for the assay of Pb content. Seedlings were grown in the air for 4 days in the presence (1 mM and 3 mM) or absence of PbCl2, as described in the above section. After the cultivation, seedlings were washed several times with deionized water, and then shoots and roots were excised. Their fresh weights were measured using an electric balance. Shoots and roots excised from the control and 1 mM Pb-treated seedlings were put in Teflon vessels and immediately oven-dried at 60 ◦C for 2 days. The dried samples were digested completely with HNO3/HClO4 (2:1, *v*/*v*) solution at 140 ◦C for 24 h. After the acidic solution was evaporated completely, the digested samples were dissolved in 0.1 N HNO3 and analyzed for Pb content using inductive coupled plasma–mass spectrometry (ICP-MS) (SPQ 9700; Hitachi High-Tech Science Corp., Tokyo, Japan). For the measurement of Pb content in cytoplasmic fluid, shoots excised from 1 mM and 3 mM Pb-treated seedlings and roots from 1 mM Pb-treated ones were boiled for 10 min in 10 mL of 80% ethanol. The ethanol extract was dried in Teflon vessels. Dried samples were digested with HNO3/HclO4 and then dissolved in 0.1 N HNO3, as described above. The Pb content in the cytoplasmic fluid of 3 mM Pb-treated roots could not be analyzed because roots of air-grown seedlings hardly grew at this concentration.

#### *2.3. Measurement of the Osmotic Concentration of Cell Sap*

The extraction and collection of cell sap were carried out according to the method of Ooume et al. [29]. The coleoptiles obtained from air-grown and water-grown seedlings were put in a plastic mini-column and then immediately frozen with liquid nitrogen. The cell sap was collected from frozen–thawed coleoptiles by centrifugation at 1500× *g* for 10 min at 4 ◦C. The osmotic concentration of the collected cell sap was measured with a vapor pressure osmometer (Model 5500C; Wescor, Logan, UT, USA).

#### *2.4. Measurement of the Mechanical Properties of the Cell Wall*

The coleoptiles prepared from air-grown and water-grown seedlings were immediately boiled for 10 min in 80% ethanol and then stored in fresh 80% ethanol. Before the measurement of cell wall mechanical properties, ethanol-fixed samples were rehydrated for several hours. Cell wall extensibility was measured with a tensile tester (RTM-25; Toyo Baldwin Co., Tokyo, Japan) [30]. The subapical region (1–2 mm below the tip) of air-grown coleoptile was fixed between two clamps 2 mm apart, and stretched by lowering the bottom clamp at a speed of 20 mm/min to produce a stress of 10 g. In the case of water-grown plants, a segment 10 mm in length was excised from the tip of the coleoptile. The segment was fixed between two clamps 2 mm apart and stretched at the same speed to produce a stress of 4 g. Cell wall extensibility (μm/g) was determined by measuring the rate of the

increase in stress just before it reached the maximum stress (4 g and 10 g for water-grown and air-grown coleoptiles, respectively).

#### *2.5. Fractionation of Cell Wall Constituents*

Cell wall materials were prepared and fractionated according to the method of Wakabayashi et al. [31]. Briefly, cell wall preparation was treated with 1 M NaOH to extract ester-linked phenolic acids. Then, the residual material was extracted with 17.5% NaOH containing 0.02% NaBH4. The fraction extracted with 17.5% NaOH was neutralized with acetic acid. After the extraction of cell wall-bound phenolic acids from the 1 M NaOH solution as described below, the remaining solution was combined with the 17.5% NaOH extracts, and designated as the matrix polysaccharide fraction. The alkali-insoluble fraction was designated as the cellulose fraction. The cellulose fraction was dissolved with 72% sulfuric acid. The total sugar content in each fraction was determined by the phenol-sulfuric acid method [32] and expressed as glucose equivalents.

#### *2.6. Determination of Cell Wall-Bound Phenolic Acids*

Analysis of cell wall-bound phenolic acids was carried out according to the method of Wakabayashi et al. [31]. Ester-linked phenolic acids liberated from the cell wall with 1 M NaOH (see above) were recovered into ethyl acetate by acidification. The liberated phenolic acids were analyzed using an HPLC system equipped with a reversed-phase column and a photodiode array detector with a gradient elution of methanol. FA and *p*-coumaric acid (*p*-CA) were identified and quantified using authentic *trans*-FA and *trans*-*p*-CA (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The peaks of DFA isomers were identified and quantified using response factors [33].

#### *2.7. Statistical Analysis*

For each measurement, the means and the standard errors of the means (SE) were calculated. The significance of differences among the treatments with different Pb concentrations was analyzed using Tukey's HSD test (*p* < 0.05). The significance of differences between the control and single Pb treatment was analyzed using Student's *t*-test (*p* < 0.05).

#### **3. Results**

#### *3.1. Effects of Pb on Air-Grown Seedlings*

When germinated rice caryopses were grown for 4 days on moistened filter paper, the lengths of primary roots and coleoptiles reached about 33 mm and 10 mm, respectively, in the absence of Pb (Figure 1). Root growth was significantly inhibited by the 0.3 mM Pb treatment and the inhibitory effect significantly increased in a concentration-dependent manner. Roots scarcely elongated at 3 mM Pb. In contrast, coleoptile growth was not inhibited by Pb up to 1 mM, but was significantly inhibited at 3 mM (Figure 1). Growth of the first leaf inside the coleoptile synchronized with that of the coleoptile until the start of leaf emergence. Pb also inhibited the growth of first leaves at 3 mM, but lower concentrations did not affect leaf growth; the lengths of first leaves on 4 days were 8.4 ± 0.3, 8.4 ± 0.2, 8.3 ± 0.3, 8.1 ± 0.2, and 4.8 ± 0.3 mm (each *n* = 18–20) for 0, 0.1, 0.3, 1, and 3 mM Pb, respectively. These results suggest that the inhibitory effect of Pb on the growth of aboveground organs in air-grown rice seedlings was much smaller than that of roots.

**Figure 1.** Effects of Pb on the growth of coleoptiles and roots of air-grown rice seedlings. Germinated caryopses were planted on filter paper containing 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. Data are means ± SE (*n* = 18–20). Different letters above the bars represent statistically significant differences (Tukey's HSD test, *p* < 0.05).

Next, the accumulation of Pb in shoots consisting of coleoptile and first leaf and in roots of air-grown seedlings was analyzed by ICP-MS. When germinated caryopses were grown for 4 days on moistened filter paper containing a buffer solution with or without 1 mM Pb, root growth was substantially inhibited by 1 mM Pb, but shoot growth was not, as shown in Figure 1. The Pb contents in the 1 mM Pb-treated shoot and root were 119 ± 25 and 1025 ± 86 ng/organ, respectively (*n* = 3), while the contents in the control seedlings were negligible (Pb contents in the control shoot and root were 0.1 and 0.4 ng/organ, respectively). We further examined the Pb accumulation in the cytoplasmic fluid of shoots and roots that had been grown for 4 days in the presence of 1 mM and 3 mM Pb. The Pb contents in the cytoplasmic fluid of the 1 mM and 3 mM Pb-treated shoots were 0.33 ± 0.07 and 3.10 ± 0.29 μg/g fresh weight (FW), respectively, while that of the 1 mM Pb-treated roots was 4.29 ± 0.18 μg/g FW (*n* = 3, respectively). The Pb content in 3 mM Pb-treated roots could not be analyzed because roots hardly grew at this concentration (Figure 1). The calculated concentrations of Pb in the cytoplasmic fluid were 1.6, 15, and 21 μM for the 1 mM Pb-treated, the 3 mM Pb-treated shoots, and the 1 mM Pb-treated roots, respectively.

Cell wall extensibility and the osmotic concentration of air-grown coleoptiles are shown in Figure 2. Treatment with Pb at a concentration of 1 mM did not affect either cell wall extensibility or the cellular osmotic concentration (Figure 2A,B), similar to the effect on coleoptile growth. The Pb treatment at 3 mM significantly decreased cell wall extensibility and increased the cellular osmotic concentration, when it inhibited coleoptile growth (Figure 1).

**Figure 2.** Effects of Pb on cell wall extensibility (**A**) and the cellular osmotic concentration (**B**) in coleoptiles of air-grown rice seedlings. Growth conditions are shown in Figure 1. (**A**) The cell wall extensibility of the upper region of coleoptiles was measured with a tensile tester. Data are means ± SE (*n* = 16–18). (**B**) The osmotic concentration of the cell sap obtained from coleoptiles was measured with a vapor pressure osmometer. Data are means ± SE (*n* = 3). Different letters above the bars represent statistically significant differences (Tukey's HSD test, *p* < 0.05).

The chemical properties of cell walls are considered to be factors determining the mechanical properties of the cell wall. We next analyzed the amounts of cell wall polysaccharides and cell wall-bound phenolic acids in air-grown coleoptiles. Cell wall polysaccharides were fractionated into two fractions, the matrix polysaccharides and cellulose. The amount of matrix polysaccharides was almost equivalent to that of cellulose in coleoptiles grown for 4 days in the air (Figure 3A). On the basis of unit length and unit fresh weight of coleoptile, Pb treatment at 3 mM significantly increased the amounts of both matrix polysaccharides and cellulose (Figure 3A).

Cell walls of gramineous plants contain phenolic acid monomers, such as FA and *p*-coumaric acid (*p*-CA). Our previous study showed that the cell walls of dark-grown rice shoots contained three predominant DFA isomers: 5-5, 8-*O*-4, and 8-5 DFA [34]. On the basis of unit matrix polysaccharide content, Pb treatment at 3 mM significantly decreased the amounts of both phenolic acid monomers, although amounts of *p*-CA were substantially lower than those of FA (Figure 3B). Furthermore, among DFA isomers, the amounts of 8-*O*-4 and 8-5 DFAs in Pb–treated coleoptiles were significantly lower than those in control ones (Figure 3B).

**Figure 3.** Effects of Pb on the amounts of cell wall polysaccharides (**A**) and cell wall-bound phenolic acids (**B**) in air-grown rice coleoptiles. Coleoptiles were grown for 4 days in the presence or absence of 3 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).
