Immunolocalization of a Normal Wood Specific Pectin Methylesterase (CoPME) and Quantification of PME Gene Expression in Differentiating Xylem of Chamaecyparis obtusa

The transverse section of compression wood tracheids has a circular shape and intercellular spaces. The cause has not been determined yet; however, we hypothesized that peeling of the cell wall adhesion would cause cellular inter-vals, resulting in circularity of the transverse section of tracheids. Homogalacturonan, a type of pectin, functions in cell wall adhesion. Further, pectin methylesterase (PME) is involved in functionalization of homogalacturonan. We quantitated PME gene expression levels in differentiating xylem cells using different degrees of compression wood samples and examined the corre-lation with circularity of the transverse section of tracheids in each sample. We found that lower gene expression level of the sample corresponded with increasing circularity of the transverse section of tracheids. It is considered that the transverse section of compression wood tracheids becomes circular by suppression of PME gene expression during differentiation. Further, we observed the normal wood specific pectin methylesterase (CoPME) localization in differentiating xylem tracheids by immunolabeling. Labels localized at the entire perimeter of the compound middle lamella in normal wood, whe-reas sparse labeling was found in compression wood. It suggests that cell walls adhere at sites of CoPME function in differentiating xylem tracheids, but there is inadequate adhesion between cell walls where CoPME

The transverse section of compression wood tracheids has a circular shape and intercellular spaces. The cause has not been determined yet; however, we hypothesized that peeling of the cell wall adhesion would cause cellular intervals, resulting in circularity of the transverse section of tracheids. Homogalacturonan, a type of pectin, functions in cell wall adhesion. Further, pectin methylesterase (PME) is involved in functionalization of homogalacturonan. We quantitated PME gene expression levels in differentiating xylem cells using different degrees of compression wood samples and examined the correlation with circularity of the transverse section of tracheids in each sample. We found that lower gene expression level of the sample corresponded with increasing circularity of the transverse section of tracheids. It is considered that the transverse section of compression wood tracheids becomes circular by suppression of PME gene expression during differentiation. Further, we observed the normal wood specific pectin methylesterase (CoPME) localization in differentiating xylem tracheids by immunolabeling. Labels localized at the entire perimeter of the compound middle lamella in normal wood, whereas sparse labeling was found in compression wood. It suggests that cell walls adhere at sites of CoPME function in differentiating xylem tracheids, but there is inadequate adhesion between cell walls where CoPME does not function. At the end of the expansion zone, the volume of the cell decreases due to a decrease in the turgor pressure of the tracheid. Further, due to moisture shrinkage of the tracheid, the adhesion begins to peel off in places of inadequate adhesion between cell walls, resulting in cell gaps and, thereby, generating a circular cell shape of cell wall formation in compression wood.

Introduction
To support inclined stems and branches, trees form specific secondary xylems called "reaction wood" in tissue morphology and physical property [1] [2]. Reaction wood for coniferous trees is called "compression wood", and they are formed on the lower sides of inclined stems and branches [2] [3] [4] [5] [6]. The formed compression wood generates large growth stress in the axial direction and bends the stem and branches growing due to growth stress to push the tree upward [7] [8]. Compression wood has different anatomical and chemical properties than normal wood [5] [6]. The main feature of compression wood is as follows: lower cellulose content and higher lignin content than normal wood, thick S1 layer, absent S3 layer, and reddish-brown color on wood surface. Furthermore, while transverse sections of normal wood tracheids forming in the vertical stems tend to have cell walls adhering to each other, the transverse sections of compression wood tracheids are circular and have intercellular spaces [3] [4] [5] [6] [9]. In this study, we attempted to elucidate the cause of compression wood tracheids becoming circular.
Intercellular adhesion in plant cells occurs via the cell wall. We examined factors involved in adhesion between cell walls and focused on the cell wall polysaccharide pectin, which plays an important role in intercellular adhesion. Pectin is the major constituent of the middle lamella and primary cell wall of plant cell walls [10] [11] [12], and it is a complex polysaccharide formed by the linking of three major structural regions of homogalacturonan (HG), rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II) [12] [13] [14] [15]. Of the pectin constituent regions, homogalacturonan is extensively methyl esterified (80% -90%) during the biosynthesis process, and it accumulates on the cell wall [11] [16] [17] [18]. Subsequently, the hydrolytic enzyme pectin methylesterase (PME, EC 3.1.1.11, CAZy CE8) catalyzes demethylation of the methyl group of homogalacturonan in the cell wall. This process releases methanol and a proton, thereby making the carboxyl group of homogalacturonan negatively charged [12] [19]. In the demethylated homogalacturonan, the negatively charged carboxyl group and Ca 2+ ions are cross-linked [17] [20]. This then promotes formation of a model structure called the "egg box" between molecules [21]  immunohistochemical staining, we examined the localization of normal wood specific pectin methylesterase (CoPME) and homogalacturonan in the differentiating xylem tracheids of Chamaecyparis obtusa. To examine the localization of CoPME, we created an antibody that specifically binds CoPME in the differentiating xylem of Chamaecyparis obtusa. Furthermore, to examine the localization of homogalacturonan, we used two types of monoclonal antibodies (LM19, LM20). They recognize different degrees of methyl esterification on homogalacturonan. LM20 recognizes highly and partially methyl-esterified (highly methylated) homogalacturonan, which is a substrate for PME. LM19 recognizes partially methyl-esterified and un-esterified (low methylated) homogalacturonan, which is a product of PME [26]. In this paper, we discuss the validity of CoPME being involved in the circularity of transverse sections of compression wood tracheid.

Plant Material
Samples for quantitative real-time PCR (qPCR) were grown from April to June 2015 in a field owned by Nagoya University, Japan. Twenty Japanese cypress (C. obtusa) saplings (about 100 cm in height) were planted in plastic pots filled with a mixture of red soil and compost. The saplings were loosely fixed to a stake using wire to maintain vertical stem growth. Four of these saplings were grown vertically to generate normal wood. In order to prepare compression wood of different levels of development, we grew 16 other saplings, with four saplings each being grown at four different tilt angles (5˚, 10˚, 20˚, and 30˚). Sampling was conducted during the most active period of cambial growth in June. After removing the bark, the stems of the saplings were cut into segments and the differentiating xylem tissues in the cut portions were harvested using a chisel. The lower sides of the stems (compression wood) were collected from the inclined American Journal of Plant Sciences saplings, and both sides of the stems were collected from the vertical saplings (normal wood). Immediately after harvesting, these tissues were frozen in liquid nitrogen and stored at −80˚C for total RNA extraction. We also prepared samples to be used for tissue observations for measuring circularity of the transverse section of the xylem tracheid. After harvesting the differentiating xylem for total RNA extraction, we sampled the stem by using a saw to cut the surface where the tilt angle of the stem was constant in each sapling.
Samples for immunolabeling and protein extraction were grown from April to June 2016. Twenty Japanese cypress (C. obtusa) saplings (about 100 cm in height) were grown at the same condition of a previous experiment. Eight of these saplings were grown at an angle (i.e. with non-vertical stems) to generate compression wood. The other twelve saplings were grown vertically to generate normal wood. Each of the saplings with compression/normal wood was used for immunolabeling. Selected pieces of stem that included differentiating compression/normal wood were harvested from the saplings and cut with a razor blade into small square blocks measuring several millimeters on each side. The other saplings with compression/normal wood were used for protein extraction. The differentiating xylem tissues were collected using the same harvesting method for total RNA extraction. Immediately after harvesting, these tissues were frozen in liquid nitrogen and stored at −80˚C for protein extraction.

Quantitative Real-Time PCR (qPCR)
Total RNA was extracted from 50 mg of differentiating xylem sample using an

Circularity Measurement of Xylem Tracheid Transverse Sections
Harvested stem segments were fixed in 3% glutaraldehyde in 67 mM phosphate buffer (pH 7.0) for 1 week at 4˚C. Subsequently, fixation in phosphate buffer was repeated. Transverse sections, 10 μm thick, were prepared from the segments using a sliding microtome. The sections were stained with 1% safranin and dehydrated in an increasing ethanol series. After soaking in xylene, the sections were mounted on glass slides with EntellanNeu (Merck, Darmstadt, Germany) and observed under a light microscope (BX60; Olympus, Tokyo, Japan). The observations were captured using a camera (DP70; Olympus, 1360 × 1024 pixels). Ten cells were selected for each sample, and the area and the perimeter of the intracellular cavity of the transverse section of the xylem tracheid were measured using the image processing software ImageJ. Following, the circularity (R) was calculated. Circularity is a parameter showing how close a shape is to a circle, and as circularity approaches 1, the closer a shape is to a true circle. Circularity is defined by the following formula: (S is area, L is the perimeter).

Determination of CoPME gene Base Sequence
To create the anti-PME antibody, we used cDNA cloning and sequencing to identify the accurate base sequence of the PME gene that was expressed in diffe-

Protein Extraction
We grinded portions (5 -10 g) of the differentiating xylem in liquid nitrogen to produce a powder. To obtain the soluble and ionically bound proteins, we ex-American Journal of Plant Sciences

Western Blotting
Rabbit anti-PME immunoglobulin G (IgG) antibody was designed based on the amino acid sequence of the PME protein (ASEGSNGNEN); the peptide of this amino acid sequence was used as the antigen to raise the rabbit IgG antibody. To absorb the anti-PME antibody into the antigen, we mixed the anti-PME anti-

Fixation and Embedding of Samples for Immunolabeling
Square stem blocks, each several millimeters on a side, were frozen rapidly by immersion in liquid chlorodifluoromethane (HCFC-22) cooled with liquid nitrogen. The blocks were transferred into an acetone solution containing 0.7% glutaraldehyde cooled to −80˚C, and then incubated for > 3 days to obtain a frozen substitute. The blocks were incubated at −20˚C overnight, then at 4˚C overnight. After equilibrating the blocks to room temperature, we immediately washed them thrice for 10 min in acetone. Subsequently, the block was immersed thrice for 1 h in 100% ethanol to replace acetone with ethanol. After this, the block was immersed overnight in a solution of ethanol:LR White resin = 1:1.

A. Ota et al. American Journal of Plant Sciences
They were embedded in LR White resin (London Resin Co., Basingstoke, UK).
The resin was cured at 50˚C overnight in airtight gelatin capsules.

Immunolabeling
Immunolabelling was conducted according to procedures described previously

Relationship between Circularity and Gene Expression Level in Transverse Sections of Differentiating Xylem Tracheid
We

Western Blotting
To determine whether the anti-CoPME antibody could specifically recognize the C. obtusa PME, we performed western blotting of the anti-CoPME antibody against proteins extracted fro/m the differentiating xylem of normal and compression wood. The anti-CoPME antibody reacted to proteins extracted from differentiating xylem of C. obtusa. We detected two signals from proteins extracted from normal wood (Figure 3(a)). A strong signal was seen in the molecular weight range of 55 and 60 kDa, while a weak signal was observed between 37 and 39 kDa. Similar results were observed from proteins extracted from compression wood (Figure 3(b)). However, the anti-CoPME antibody absorbed by antigen did not react to proteins extracted from the differentiating xylem of C. obtusa (Figure 3(a), Figure 3(b)).

Immunolocalization of PME in Differentiating Xylem Tracheid Using Anti-CoPME Antibody
The anti-CoPME antibody was used for immunolabeling to observe the transverse   . Specificity of the anti-PME antibody by Western blotting. (a) Reaction of proteins from normal wood with the anti-PME detected one strong signal and one weak signal (four lanes on the left side). Reaction with anti-PME antibody absorbed with antigen did not produce signals (four lanes on the right side); (b) Similar results of proteins from normal wood were obtained from compression wood proteins reacted with anti-PME antibody (four lanes on the left side) and anti-PME antibody absorbed with antigen (four lanes on the right side).
section of differentiating xylem tracheid from the cambium to the xylem tracheid. In sections of normal wood, labeling was seen in the compound middle lamella from the cambium to the expansion zone (Figure 4(b)). There was labeling across the entire perimeter of the cell wall after the expansion zone ended and thickening of the secondary wall started. There was also labeling in the cell corner. Furthermore, in sections of compression wood, there was sparse labeling on the cell walls from the cambium to the mature cell ( Figure 5(b)). From the

Immunolocalization of Homogalacturonan in Differentiating Xylem Tracheid Using LM19 and LM20 Antibodies
The anti-homogalacturonan antibodies LM19 and LM20 were used for immunolabeling to observe the transverse section of differentiating xylem tracheid from the cambium to the xylem tracheid. LM19 recognizes low methylated homogalacturonan, while LM20 recognizes highly methylated homogalacturonan.
Observation of normal wood sections using the LM19 antibody showed labeling in the compound middle lamella from the cambium to the expansion zone ( Figure 6(b)). There was particularly intense labeling in the radial wall. When the LM20 antibody was used to observe normal wood sections, there was labeling in the middle lamella between the cambium and expansion zone ( Figure 8(b)). There was particularly intense labeling in the radial wall. After the end of the expansion zone and start of the thickening of secondary wall, there was labeling in the radial wall, but labeling in the tangential wall had weakened.
There was intense labeling in the cell corner. There was no labeling in the secondary wall at the stage when thickening had completed. Similar labeling patterns as normal wood sections were seen in compression wood sections ( Figure   9(b)). Moreover, using the LM20 antibody absorbed with 1 mg/mL pectin solution, neither normal wood nor compression wood sections showed labeling ( Figure 8(d), Figure 9(d)). Neither normal wood nor compression wood sections showed any labeling using a control that had LM19 and LM20 removed ( Figure 6(f), Figure 7(f)).

Relationship between Circularity in the Transverse Section of xylem Tracheid and Gene Expression Levels
The graph, which shows the relationship between relative PME gene expression

Test of the Anti-CoPME Antibody Specificity
We used Western blotting to test the specificity of the anti-CoPME antibody. In the normal wood sample and compression wood sample, we detected a strong signal near molecular weights of 55 -0 kDa. We converted the cDNA sequence of the PME gene functioning in the differentiating xylem, obtained by sequencing, to an amino acid sequence and analyzed the sequence using the EX-Pazy-ProtParam tool (https://web.expasy.org/protparam/). This revealed that the total molecular weight of this protein was at least 53 kDa. Therefore, this suggests that the anti-CoPME antibody may have recognized and bound to the PME present in the differentiating xylem.
Furthermore, in both the normal wood sample and compression wood sample, we detected a signal near molecular weights of 37 -39 kDa. We believe that this signal recognized the PME cleaved at a certain site in the amino acid region by proteases. However, it is also possible that the anti-CoPME antibody is non-specifically bound to a different protein with a similar sequence to the peptide sequence (ASEGSNGNEN). In order to identify the protein detected by the anti-CoPME antibody, it is necessary to recover the protein bound to the anti-CoPME antibody and investigate the amino acid sequence of said protein.
However, anti-CoPME antibody absorbed by antigen did not recognize any protein extracted from the differentiating xylem. For this reason, we believe that the signal detected was due to recognition of PME present in the differentiating xylem by the anti-CoPME antibody.

Localization of CoPME in the Differentiating Xylem Tracheids
Through immunolabeling using the anti-CoPME antibody, we examined CoPME localization in transverse sections of the differentiating xylem tracheids. CoPME labeling was observed in the compound middle lamella between the cambium and expansion zone in sections of normal wood and compression wood. Labeling was also mainly observed in the compound middle lamella, from the expansion zone to the mature cell. As a result, we inferred that CoPME was present in the compound middle lamella between the cambium and mature cells. It has been shown that in the differentiating xylem of Populus euramerica branches, PME is present in the compound middle lamella of the radial wall and in the cell corner [35]. This study produced similar results. When immunolabeling was carried out using anti-CoPME absorbed with antigen, there was no labeling. Therefore, we believe that the labeling indicated CoPME recognition present in the differentiating xylem tracheids by the anti-CoPME antibody.

Localization of Homogalacturonan in the Differentiating Xylem Tracheids
Through immunolabeling using the anti-homogalacturonan antibodies LM19 and LM20, we examined homogalacturonan localization in transverse sections of the differentiating xylem tracheids. LM20 recognizes highly methylated homogalacturonan, which serves as a substrate when PME is functioning. LM19 recognizes low methylated homogalacturonan, which is produced when PME functions.
LM20 labeling was observed in compound middle lamella between the cambium and expansion zone, in both normal wood and compression wood sections. Labeling was also observed in compound middle lamella of the radial wall, from the expansion zone to the mature cell. This result suggested that highly methylated homogalacturonan, which is the substrate when PME is functioning, is present in abundance in the compound middle lamella between the cambium and mature cell. In the differentiating xylem of Populus euramerica branches, highly methylated homogalacturonan is reportedly present in the middle lamella of the radial wall and tangential wall in the cambium [34] [36], and similar results were observed in this study. No labeling was observed when immunolabeling was performed using the LM20 antibody absorbed with pectin solution.
Therefore, we believe that labeling showed recognition of highly methylated homogalacturonan present in the differentiating xylem tracheids by the LM20 antibody. LM19 labeling was observed in compound middle lamella between the cambium and expansion zone in both normal wood and compression wood sections. The intensity of labeling weakened from the expansion zone to the mature cell.
In the differentiating xylem of Populus euramerica branches, low methylated homogalacturonan is reportedly weakened significantly as the secondary wall was thickening, and similar results were observed in this study [36]. LM19 antibody may not have recognized the low methylated homogalacturonan, which composed the pectin gel that functions as an adhesive between cell walls. In the differentiating xylem of Populus euramerica branches, low methylated homogalacturonan is reportedly present in the intermediate layer of the radial wall in the cambium [34], but this study did not produce similar results. No labeling was observed when immunolabeling was performed using the LM19 antibody absorbed with polygalacturonic acid solution. Therefore, we believe that labeling indicated recognition of low methylated homogalacturonan present in the differentiating xylem tracheids by the LM19 antibody.
It is also conceivable that highly methylated homogalacturonan is present in greater quantity than low methylated homogalacturonan in the cell walls of ma-ture cells. In mature cells, highly methylated homogalacturonan may be the majority form of homogalacturonan present, and low methylated homogalacturonan may only be present in small amounts. However, the result in this study, in which far more labeling of highly methylated homogalacturonan than low methylated homogalacturonan in the compound middle lamella was found, was similar to the observations made from immunohistochemical staining of homogalacturonan in mature xylem of Pinus sylvestris [37]. Several hypotheses have been proposed to explain the greater amount of highly methylated homogalacturonan than low methylated homogalacturonan in cell walls after lignification. It has been reported that the presence of Ca 2+ , which is said to crosslink the carboxyl groups in low methylated homogalacturonan, causes one-electron oxidation of coniferyl alcohol and is involved in the lignification process [38]. In other words, the liberation of Ca 2+ from homogalacturonan might be necessary for lignification to start after the end of the expansion zone. It has also been reported that in the presence of Ca 2+ , low methylated homogalacturonan has affinity to isoperoxidase, which is responsible for polymerization of lignin [39]. These reports suggested that liberated Ca 2+ and low methylated homogalacturonan may be involved in lignification of the cell wall.

Difference in Localization of CoPME between Tracheid of Normal Wood and Compression Wood
When the distribution of localized CoPME was compared between normal wood and compression wood sections, we observed that normal wood had more intense labeling than compression wood in the compound middle lamella from the cambium to expansion zone. This result suggested that the amount of CoPME present in the differentiating xylem of normal wood was greater than compression wood. This result also supports the relationship between increased circularity of transverse sections of the tracheid and decreased PME expression levels in Figure 10. Schema for the hypothesis of generating a circular cell shape. In normal wood, CoPME labels localized at the entire perimeter of the compound middle lamella. In compression wood, their labels localized at sparse on the cell walls. It suggested that cell walls adhere at sites of CoPME function in differentiating xylem tracheids.
differentiating xylem cells. Further, we observed labeling around the entire perimeter of the cell wall in normal wood sections. Conversely, compression wood sections did not show labeling around the perimeter of the cell wall and, instead, showed sparse labeling. This result suggested that while CoPME was present all around the cell wall of normal wood, the same was not true for compression wood. We believe that cell shape becomes circular according to the following process of cell wall formation in compression wood. In places where CoPME functions, pectin gel formation allows sufficient adhesion between cell walls.
However, in places where CoPME does not function, we believe that there is inadequate adhesion between cell walls. At the end of the expansion zone, the volume of the cell decreases due to a decrease in the turgor pressure of the tracheid. Further, due to moisture shrinkage of the tracheid, the adhesion begins to peel off in places of inadequate adhesion between cell walls, resulting in cell gaps and, thereby, generating a circular cell shape ( Figure 10).