Decrease in cell size and cell wall components under ectopic expression of the CaRLK1 gene

The Capsicum annuum receptor-like kinase 1 ( CaRLK1 ) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain. It functions as a negative regulator of plant cell death. Ectopic expression of CaRLK1 showed the hypoxia-resistance and enhanced cell division and proliferation. In this study, it was investigated which genes were controlled by ectopic expression of CaRLK1 because it decreased its average cell size of transgenic RLK1ox cells compared with that of wild-type cells (BY-2). glucomannan-4-beta-mannosyltransferase genes were suppressed. The polygalacturonase genes may also contribute to make the cell wall of RLK1ox cell thinner than that of BY-2 cell. Of special emphasis is its impact of CaRLK1 gene on cell size control and cell wall thickness. Smaller cell size of the RLK1ox cells correlates with the inhibited cell expansion.


Background
It is known that cell size is related to the physiological state of the plant cell [1,2]. Therefore, the cell size distribution directly reflects the average physiological properties of the suspension cell culture. Cells need to be small because they need a high 'surface to volume' ratio, which is good for exchanging materials (ions, organic molecules, and waste) between the inside and outside of cells.
The plant cell wall, as a dynamic structure, plays important roles in controlling the differentiation of plant cells during embryogenesis and growth, including cell enlargement, cell proliferation, and fruit softening. The primary cell wall of land plants is composed of the polysaccharides cellulose, hemicelluloses and pectin [3][4][5]. Cellulose is an important structural component of the primary cell wall of green plants and plays a central role in determining the mechanical properties of plant cell walls. Cellulose is an organic compound consisting of a linear chain of glucose units. Hemicellulose is one of a number of heteropolymers (matrix polysaccharides), including xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. Thus, hemicelluloses include the five-carbon sugars xylose and arabinose, the six-carbon sugars glucose, mannose and galactose, and the six-carbon deoxy sugar rhamnose.
Glucomannan is a water-soluble polysaccharide that is considered a dietary fiber. This polysaccharide acts as a hemicellulose component in the cell walls of some plant species. Pectin is a highly hydrated network of structural acidic heteropolysaccharides rich in galacturonic acid.
Pectin is mainly found in the middle lamellae in the primary cell walls of virtually all terrestrial plant cells. In primary cell walls, the matrix in which the cellulose network is embedded is composed of pectin.
Cellulose is synthesized at the plasma membrane by a large multiprotein complex, known as the cellulose synthase complex (CSC) [6,7]. CSCs contain multiple nonredundant cellulose synthase A (CESA) proteins that serve as CSC catalytic subunits. The CESA1, CESA3, CESA6, and CESA9 genes are involved in primary cell wall cellulose biosynthesis [7].
Cellulose synthesis in secondary walls involves three functionally nonredundant secondary wall CESAs belonging to the glycosyltransferase family 2 (CESA4, CESA7, and CESA8).
During organ development, plant cells can be enlarged considerably by the expansion process, where water accumulates internally through osmosis and plant cells have a rigid cell wall surrounding the plasma membrane [5]. Such hormones as auxins, gibberellins, ethylene, and cytokinins direct the coordinated growth and proliferation of plant cells. Expansins [8] and polygalacturonase (PG) [9] are known to be responsible for loosening the structure of the wall.
Expansins (EXP) were discovered to expand growing plant cell walls faster at low (acidic) pH than at neutral pH and to influence the structure of cellulose/hemicellulose frameworks. The mRNA levels of expansin genes and expansin protein levels are strongly correlated with the growth and development of root and shoot organs [10,11]. The expression of CaEXPA14 and CaEXPA19 positively correlated with the rate of cell expansion in bell pepper [12]. Expansins have been shown to bind strongly to cellulose and to allow loosening of the wall by disrupting the hydrogen bonds. Expression of both EXPAs and EXPBs was associated with internode elongation in deep-water rice [13]. Expansins are wall-loosening proteins implicated in plant responses to most of the major plant hormones, such as auxin [8], gibberellin (GA) [14], cytokinin [15], and ethylene [16], in several organs and species. AtEXP8 and AtEXP18 in Arabidopsis [17] and OsEXP1 and OsEXP2 in rice [18] are responsive to ethylene. Four expansins (EXPA4, EXPA5, EXPA7, and EXPB4) are upregulated by GA treatment during GA stimulation of leaf elongation in Festuca arundinacea [19]. Submergence-induced petiole elongation enables R. palustris to emerge from flooded environments. Ethylene promotes the transcription of an expansin gene (RpEXPA1) in R. palustris [20,21]. The Brassica rapa expansin-like B1 gene (BrEXLB1) regulates growth and development in transgenic Arabidopsis and elicits responses to abiotic stresses. Overexpressors of sense BrEXLB1 enhance plant height [33].
Polygalacturonase (EC 3.2.1.15) is an enzyme that hydrolyzes the α-1,4 glycosidic bonds between galacturonic acid residues. This enzyme is a pectinase that degrades pectin to soften and sweeten fruit. Ethylene increases PG, which degrades pectin to loosen the cell wall.
ACC (1-aminocyclopropane-1-carboxylate) synthase (ACS) catalyzes the rate-limiting step of ethylene biosynthesis [22]. ACS6 (At4g11280) was unregulated by ABA, BL, and IAA. ACS10 (At1g62960) was downregulated by ABA, IAA, MJ, and GA. The activities of GA 20-oxidases (GA20ox), GA 3-oxidases (GA3ox), and GA 2-oxidases (GA2ox) are of particular importance in determining gibberellin (GA) concentration in many plant species [23,24]. Cellular accumulations of gibberellins regulate cell growth in developing embryos, elongating stems and roots, and developing floral organs. GA2ox enzymes catalyze the main inactivation step through 2-oxidation of both the bioactive forms and their precursors. Thus it can inhibit cell expansion by reducing the concentration of the bioactive GAs.
Tobacco BY-2 cell (Nicotiana tabacum) has been employed as a model cell in various aspects in plant physiology [25]. BY-2 cell is very useful to investigate the dynamics of a single type of plant primary cell wall.
The functions of CaRLK1 gene (Capsicum annum receptor-like kinase1) isolated from hot pepper [26] are associated with suppressing plant cell death, hypoxia resistance, and promoting cell division [27,28]. The amino acid sequences of CaRLK1 gene are very similar to leaf rust resistance kinase Lr10 genes of the Solanaceae family including pepper, tomato and tobacco. In the previous result, overexpression of CaRLK1 gene promotes cell division with decreased cell size [28]. Thus, we are very interested in elucidating how ectopic expression of the CaRLK1 gene regulates cell size and cell wall thickness. In this study, the overall goal was achieved to elucidate the biological and genetic mechanisms; which genes are highly regulated under ectopic expression of the CaRLK1 gene.

Cell size distribution
In general, eukaryotic cells have diameters ranging from 10 to 100 μm. As a cell decreases in size, its surface area-to-volume ratio increases. Ectopic expression of the CaRLK1 gene in N. tabacum BY-2 cells decreased cell size: The average diameter of the RLK1ox protoplasts was smaller than that of the protoplasts (named as BYK) to that the control vector was introduced [28]. The AD and diameter distribution (DD) of the protoplast were chosen as the evaluation criteria for the cell size test. A detailed analysis of representative protoplasts showed that the AD of RLK1ox cells was 30.53±0.37 µm on average, whereas the AD of BY-2 control cells was 41.38±0.40 µm (n≥500) (Fig. 1A). The BY-2 cells grew to an average volume of 37,081 μm 3 , whereas the RLK1ox cells grew to an average volume of 14616 μm 3 , indicating that the size of RLK1ox cells is considerably smaller than that of BY-2 cells. Thus, the cell volume of RLK1ox cells also decreased more than 2.5-fold. In addition, the BY-2 cells grew to an average surface area of 5377 μm 2 . However, the RLK1ox cells grew only to an average surface area of 2890 μm 2 (data not shown).
A variation in DD was observed when 7-day-old cells were compared (Fig. 1B). The DDs of BY-2 cells and RLK1ox cells were not similar to each other. In the range of 10-50 µm, the DD in RLK1ox cells is over 85%, whereas the DD in BY-2 cells is less than 43%. This result indicates that the small cell size of RLK1ox cells is considerably more than that of BY-2 cells.
Cells need to be small because they need a high 'surface to volume' ratio, which enables them to rely on oxygen and material diffusing into the cell and waste diffusing out to survive and grow. Thus, smaller RLK1ox cells have a higher surface area to volume ratio (1.21fold) than larger BY-2 cells, indicating that RLK1ox cells are better for exchanging materials (ions, organic molecules, oxygen, and waste) between the inside and outside of cells than BY-2 cells.

Thin cell walls
Ectopic expression of the CaRLK1 gene in a Nicotiana tabacum BY-2 background promotes proliferation at an approximately 3-fold higher rate than wild-type BY-2, either in suspension culture or on solid medium [28]. Thus, we hypothesized that the cell walls of fastdividing RLK1ox cells are thinner than those of relatively slow-dividing BY-2 cells.
The thickness of the cell walls was measured by transmission electron microscopy (TEM). The thickness of a double cell wall layer between two RLK1ox cells appeared narrower than that between two BY-2 control cells ( Fig. 2A and 2B). The TEM images in Figure 2B reveal representative images of a double cell wall layer of either BY-2 cells or RLK1ox cells.
The thickness of a double cell wall layer of the transformed cells (RLK1ox cell) is, on average, 138.83±4.12 nm, whereas that of BY-2 control cells was 156.58±4.54 nm ( Fig. 2A). In addition, the cell walls of a single layer in the RLK1ox cells were also considerably thinner than those in the BY-2 control cells (Fig. 2C). The thickness of a single cell wall layer of the RLK1ox cells is, on average, 102.79±2.68 nm, whereas that of BY-2 control cells was 171.55±5.58 nm (Fig. 2C). Figure 2D presents a representative TEM image showing the cell wall thickness of a single layer of BY-2 cells and RLK1ox cells. These observations indicate that the cell wall of RLK1ox cells is clearly thinner than that of BY-2 cells. These results suggest that cellulose biosynthesis in the RLK1ox cell is relatively inhibited than in the BY-2 cell. Generally, the advantage of thinner cell wall is that substances, such as oxygen, organic molecules, and ions, can diffuse easily into the cell whereas wastes can diffuse out easily out of cell. This result suggests that RLK1ox cell is better to survive and to proliferate than BY-2 cell.

Neutral sugar contents of the cell wall
Differences in wall thickness may be due to differences in the amount of cell wall components [29]. The amount of 6 neutral sugars from cell walls was quantified (glucose, galactose, mannose, xylose, arabinose, and rhamnose). The total neutral sugar content of purely extracted cell walls was lower in the RLK1ox cells than in the BY-2 control cells (n=3). The sum of neutral sugars in the wild-type cells constituted approximately 22.3% of the total weight of the extracted cell wall, whereas the sum of average neutral sugars in the RLK1ox cells made up only 17.3% of the total weight of the extracted cell wall (Fig. 3).
The contents of glucose, mannose, arabinose, and xylose were consistently lower in the RLK1ox cells than in the wild-type cells (Fig. 3). This result suggests that the biosynthesis of cellulose and hemicellulose polysaccharides (xylans, xyloglucans, mannans and glucomannans) are defective in fast-dividing RLK1ox cells.
This observation suggests that both the biosynthesis and incorporation of cell wall components is relatively slower in RLK1ox cells than in BY-2 cells, indicating a dynamic process of cell wall biosynthesis that is tightly downregulated during cell growth in RLK1ox cells. Plant cells surround themselves with a cell wall that greatly limits exchange materials with the extracellular world. Thus, the thinner cell walls

Expression of CESA genes
We used a Nimblegen Nicotiana tabacum 135K microarray (GreenGene Biotech, Seoul, ROK) to evaluate the upregulation and downregulation of N. tabacum genes. CESA genes are believed to encode the catalytic subunit of plant cellulose synthase. It is important to investigate whether the expression of tobacco CESA genes is changed by introducing the

CaRLK1 gene into BY-2 cells because the cell wall is thinner in RLK1ox cells and the neutral sugar content of the cell walls is decreased in RLK1ox cells compared to BY-2 cells.
A total of 14 CESA genes were detected in the microarray analysis. The p-value of eight genes among them was lower than 0.05. The expression levels of seven genes were decreased in RLK1ox cells, except for the CESA8 gene (Fig. 4). Although the expression of CESA8 (cellulose synthase A catalytic subunit 8) appears to be strongly induced in RLK1ox cells, its expression level is notably low. CESA8 is known to be involved in the biosynthesis of the plant secondary cell wall (SCW). The expression of six CESA genes (three CESA1, two CESA3, and one CESA9) is relatively high in BY-2 cells. However, the expression of these genes is suppressed in RLK1ox cells (Fig. 4). These six genes are known to be involved in the biosynthesis of the plant primary cell wall (PCW). The suppressed expression of the CESA1, CESA3, and CESA9 genes may affect the biosynthesis of the primary cell wall in RLK1ox cells.
This observation suggests that decreased expression of six CESA genes may be associated with a decrease in cell wall thickness and neutral sugar content in RLK1ox cells.

Expression of glucomannan 4-β-mannosyltransferase genes
Glucomannan 4-β-mannosyltransferase (or glucomannan synthase, CSLA9, GMMT) is an enzyme that catalyzes the biosynthesis of glucomannan, a hemicellulose component, using GDP-mannose and glucomannan polymers. A total of six GMMT genes were detected in the microarray analysis. The p-value of these genes was lower than 0.008. The expression levels of 6 genes were determined to be decreased in RLK1ox cells (Fig. 5A). In particular, three genes (Ntabacum_ESTC011116; GMMT2-1, Ntabacum_ESTC000635; GMMT2-2, and Ntabacum_ESTC000636; GMMT2-3) that are relatively highly expressed in BY-2 cells are suppressed in RLK1ox cells. In addition, mannose-1-phosphate guanylyltransferase catalyzes the biosynthesis of GDP-mannose, a substrate for glucomannan 4-β-mannosyltransferase. The expression of three genes among the 4 detected mannose-1-phosphate guanylyltransferase genes slightly decreased in RLK1ox cells (Fig. 5B). These results suggest that the suppression of two kinds of genes may decrease the mannose and glucose content of hemicellulose in the primary cell wall of RLK1ox cells.

Expression of polygalacturonase (PG) genes
The developmental changes during tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG) [9,30]. Pectin are major constituents of primary cell walls in eudicots. PGs, as pectinases, are key homogalacturonan-hydrolyzing enzymes that function in a wide range of developmental processes. PG genes were detected in microarray analysis. The p-value of seventeen PG genes among them is lower than 0.05. The expression levels of fourteen PG genes decrease in RLK1ox cells. The expression levels of 10 PG genes among 14 genes are decreased more than 2 times in RLK1ox cells than in BY-2 cells (Fig. 6). The constitutive suppression of PG in RLK1ox cells may provide a phenocopy of cell wall-deficient transgenic cells for defining the functions of the RLK1ox cells, resulting in thin cell walls.

Expression of GA2ox genes
Gibberellins (GAs) are plant hormones that regulate various developmental processes, including stem elongation, germination, dormancy, flowering, and flower development. In this study, we showed that 7 different GA2ox genes are induced in RLK1ox cells compared with control BY-2 cells (Fig. 7A). A total of 9 GA2ox genes were detected in the microarray analysis.
The p-value of 7 genes among them is lower than 0.012. The expression levels of these 3 GA2ox genes (GA2ox2, GA2ox3-1, and GA2ox4) are relatively strongly induced in RLK1ox cells, whereas the expression levels of the other 4 genes are slightly induced. The expression of two genes (GA2ox2 and GA2ox3-1) is highly induced by qRT-PCR ( Fig. 7B and 7C). The highly induced GA2ox genes may decrease the active gibberellin concentration in RLK1ox cells.
These data suggest that a low concentration of active GAs is correlated with smaller cells and negative cell expansion in RLK1ox cells. However, the expression of two GA20ox genes (GA20ox1; Ntabacum_ESTC018089 and GA20ox2; Ntabacum_ESTC015362) and one GA3ox (Ntabacum_ESTC021120) gene is not induced in RLK1ox cells (data not shown), suggesting that GA2ox genes is related with small cell size. The p-value of these 3 genes is higher than 0.18.

Expression of expansin genes
Expansins are known to be cell wall proteins that mediate acid-induced growth by catalyzing the loosening of plant cell walls without lysis of wall polymers [3]. Cell elongation is controlled by cell extensibility, which is regulated by cell wall-loosening proteins and enzymes, including expansin and xyloglucan endotransglycosylase (XET) [31]. It was reported that EXPA4 was highly expressed in germinating seeds and that EXPA3 was highly expressed in young roots and germinating seeds in tobacco [32].
A total of 28 expansin A (EXPA) were detected in the microarray analysis. The p-value of sixteen genes among them is lower than 0.015. The expression of these genes is inhibited in the RLK1ox cells, except for four genes (EXPA6-2, EXPA10-1, EXPA11-2, and EXPA12), which are slightly induced (Fig. 8A). Genome-wide gene expression analysis showed that the expression of six EXPA genes is strongly suppressed more than twofold in RLK1ox cells. In particular, the expression of EXPA3-1 (Ntabacum_ESTC008617) and EXPA4 (Ntabacum_ESTC004061) is significantly suppressed in RLK1ox cells. qRT-PCR analysis confirmed that the expression of the EXPA4 gene is suppressed approximately 2-fold and that the expression of the EXPA11-1 gene is strongly suppressed approximately 5-fold in RLK1ox cells compared to BY-2 cells (Fig. 8B, 8C).
Overexpressors of sense BrEXLB1 enhance plant height compared with wild-type plants [33]. The EXLB1 gene was reported to be highly expressed in young roots and mature roots [32]. A total of 7 expansin-like B1 (EXLB1) genes were detected in the microarray analysis. The p-value of six genes among them is lower than 0.029. Genome-wide gene expression analysis showed that the expression of all six EXLB1 genes is strongly suppressed more than twofold in RLK1ox cells (Fig. 8D). In particular, the expression of EXLB1-4 (Ntabacum_ESTC017701), which is highest among the six EXLB1 genes in BY-2 cells, is significantly suppressed in the RLK1ox cells (<3.65-fold). qRT-PCR analysis confirmed that the expression of the EXLB1-4 gene is suppressed approximately 2-fold in RLK1ox cells compared to BY-2 cells (Fig. 8E). qRT-PCR analysis confirmed that the expression of the EXLB1-4 gene is strongly suppressed by approximately 9-fold in RLK1ox cells compared to BY-2 cells.
However, a total of 6 expansin-like A genes (EXLA) were detected in the microarray analysis. The p-value of three EXLA2 genes among them is lower than 0.001 (Table S2).
However, the expression of these genes increases slightly in the RLK1ox cells (their fold change is less than 1.68), and their expression levels are relatively high in BY-2 and RLK1ox cells (Table S2). These results suggest that 3 EXLA2 genes may play important roles in cell division, rather than cell elongation, in RLK1ox cells.

Expression of ethylene biosynthesis genes
ACC synthase (ACS) is the key regulatory enzyme in the ethylene biosynthetic pathway, and ACC oxidase (ACO) converts 1-aminocyclopropane-1 carboxylic acid (ACC) to ethylene [22]. A total of 7 ACS genes were detected in the microarray analysis. The p-value of the three genes among them is lower than 0.005. The expression of two genes (Ntabacum_ESTC015881 and Ntabacum_ESTC002216) decreases in the RLK1ox cells (their fold change was more than 1.90) (Fig. 9A). In particular, the steeply decreased expression of the Ntabacum_ESTC002216 gene may contribute to the decrease in ethylene concentration in the RLK1ox cells because it is expressed at the highest level among the 3 genes in BY-2 cells.
qRT-PCR analysis confirmed that the expression of the ACS (Ntabacum_ESTC002216) gene is greatly suppressed approximately 12-fold in RLK1ox cells compared to BY-2 cells (Fig. 9B).
However, the expression level of one gene (Ntabacum_ESTC004582) is notably low in both BY-2 cells and RLK1ox cells and is slightly induced in RLK1ox cells (its fold change is less than 1.60).
A total of 17 ACO genes were detected in the microarray analysis (Fig. 9C). The p-value of nine genes among them is lower than 0.033. The expression levels of seven genes are notably low in both BY-2 cells and RLK1ox cells. However, the expression level of one ACO gene (Ntabacum_ESTC020923) is highest in BY-2 cells but decreases by 2.6-fold in RLK1ox cells.
qRT-PCR analysis confirmed that the expression of Ntabacum_ESTC020923 is strongly suppressed by more than 53-fold in RLK1ox cells compared to BY-2 cells (Fig. 9D). Sadenosyl-l-methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene in plants. SAM synthase is involved in the synthesis of SAM. The expression levels of 4 SAM synthase genes are not notably changed and show no significant regulation of ethylene biosynthesis (Fig. S4). Thus, ethylene biosynthesis in RLK1ox cells is decreased, primarily due to suppression of the expression of both ACO genes and ACS genes, rather than SAM synthase expression.

Discussion
Our results show that ectopic expression of the CaRLK1 gene negatively regulates cell size and cell wall biosynthesis based on analyses of (1) smaller protoplast size; (2)  Cell wall thickness and the amount of cell wall components were investigated for a possible explanation for the smaller protoplast size of RLK1ox cells. Cellulose is synthesized at the plasma membrane by a large protein complex, known as the cellulose synthase complex (CSC). The primary wall CESA complex consists of three CESA subunits, namely, CESA1, CESA3, CESA6, and CESA9 [34]. In the RLK1ox cells, the expression of three kinds of primary cell wall CESA complex genes (two CESA1 genes, two CESA3 genes, and two CESA9 genes) was also downregulated (Fig. 4). Sucrose synthase 1 (SUS1) is not only the source of UDPglucose for cellulose synthesis but also part of a large soluble catalytic domain of the CESA complexes, which are involved in cellulose production by channeling UDP-Glc to the catalytic subunits of the CSC [35,36]. The expression of all 5 SUS1 genes was decreased in RLK1ox cells (Fig. S1). Low expression of SUS1 genes can decrease cellulose biosynthesis in the primary cell wall due to the reduction of UDP-Glc, a substrate for CSCs. These observations can explain how lower amounts of both primary cell walls and neutral sugar contents occurred in RLK1ox cells compared with BY-2 cells.
Expression of a WEE1 gene (Ntabacum_ESTC006562) increases more in RLK1ox cells than in control cells in 0-to 3-day-old culture (Fig. S2) and in the microarray analysis (Table S2). This result suggests that upregulation of WEE1 gene under the control of CaRLK1 gene makes RLK1ox cells smaller than BY-2 cells, as both constitutive and induced expression of Arath; WEE1 in BY-2 cultures results in a reduction in mitotic cell size [37]. In addition, in RLK1ox cells the suppressed expression of both ACS genes and ACO genes may contribute to decreased ethylene biosynthesis (Fig. 9) and may not have a positive effect on inducing cell expansion through stimulation of expression of Expansin11 gene, such as in petioles [38]. A negative regulator of the ethylene response pathway, serine/threonine-protein kinase Constitutive triple response1 (CTR1), plays an important role in cell elongation in Arabidopsis [39]. Only one gene of CTR1 (Ntabacum_ESTC012515) was detected, and its expression is suppressed approximately 4.59-fold in RLK1ox cells compared to BY-2 cells (Table S2). This suppressed expression of the CTR1 gene may contribute to reducing cell elongation and cell size in RLK1ox cells.
Cell wall-modifying proteins, including PGs and EXPs, cooperatively disassemble wall polysaccharide networks in tomato fruit. The pg/exp fruit were observed to be firmer and had denser cell walls than fruit of the other genotypes [9]. Recently, the endo-PG Polygalacturonase involved in expansion1 (PGX1) was shown to be involved in hypocotyl elongation and cell expansion [40]. Lower expression of PG genes may be associated with the fact that RLK1ox cells are smaller than BY-2 cells.
Expansins, as cell wall proteins, mediate acid-induced growth by catalyzing the loosening of plant cell walls without lysis of cell wall polymers. In other words, expansins have an essential role in cell wall-loosening activity to promote plant cell elongation and expansion under various conditions by binding to complex cell walls. RLK1ox cells proliferate more quickly than BY-2 cells [27,28]. Decreases in the expression of 6 EXPA genes and 6 EXLB genes indicate that the RLK1ox cells are less expandable and elongated than the BY-2 cells, and that the cell wall thickness of the RLK1ox cells is narrower than that of the BY-2 cells (Fig.   8). This result indicates why RLK1ox cells are smaller than BY-2 cells. In addition, we suggest that the decreases in the expression of both EXP genes and PG genes cause the cell walls of RLK1ox cells to be thinner than those of BY-2 cells (Fig. 1, 2, and 6).
The simultaneous suppression of PG genes, EXPA genes, and EXPB1 genes can reduce cell wall disassembly; RLK1ox cells are more compact, have fewer cell wall components and neutral sugar contents, and have thinner cell walls than BY-2 cells (Fig. 2 and 3). It is not excluded the possibility that the protoplasts collections may have different distributions of cell cycle stages and this may account for the differences in cell size because cell doubling time was affected by ectopic expression of CaRLK1 gene [27].

Conclusion
Ectopic expression of the CaRLK1 gene make cell size smaller and cell wall thinner.
Smaller cell size of the RLK1ox cells is related with suppressed cell expansion. It was showed that exogenous expansins induced BY-2 cell growth (cell expansion) 3-fold [41]. However, ectopic expression of the CaRLK1 gene suppressed not only the expression of 12 EXPA genes and 6 EXLB genes but also that of 2 ACS genes and 5 ACO genes. But the highly induced expressions of 7 GA2ox genes can inactivate precursor and active gibberellins in the RLK1ox cell, suggesting that ectopic expression of the CaRLK1 gene suppress cell growth and expansion. These results support the fact that the RLK1ox cell is less expandable than the BY-2 cell.
A smaller cell is more effective and transporting materials, including oxygen, nutrients, and waste products, than a larger cell. That is, the larger a cell gets, the more difficult it is for nutrients and gases to move in and out of the cell. Thus, RLK1ox cells can better take up sufficient nutrients and oxygen to service their cell volume than wild-type, because their cell size is smaller and their cell wall is thinner than BY-2 cells'.
We postulate that RLK1ox cells may compete in producing proteins more than wildtype cells based on such characteristics as smaller cell size, fast dividing characteristic, and powerful proliferation because it is well known that BY-2 cells can be used as host cells for the large-scale production of recombinant proteins [45].

Cell Cultures and Growth Conditions
Wild-type and two transgenic Nicotiana tabacum L. cv Bright Yellow 2 (BY-2) cell lines were mainly used in the studies [27].  [27,28]. Wild-type BY-2, transgenic BYK vector control cell, and transgenic tobacco cell lines (RLK1ox) were also used to conduct the studies [28]. These calli were maintained in solid media at 25 ℃ in the dark and were subcultured every two weeks into fresh solid culture media. Most of the experiments were performed using cells in exponential growth phase.

Transmission electron microscopy (TEM)
For TEM analysis to measure cell wall thickness [42], suspension cells were grown for 7 days at 25℃, and samples were taken and fixed with a mixture of fresh 2% glutaraldehyde (v/v) and 2% paraformaldehyde (v/v) in 50 mM cacodylate buffer (pH 7.2) at RT for 4 h. After washing, the samples were postfixed with 1% osmium tetroxide in 50 mM cacodylate buffer at RT for 1 h. After serial dehydration with ethanol, the samples were infiltrated with a mixture of ethanol and London Resin White (LR White) (London Resin Co., London, UK). After substitution with an ascending LR White series, the samples were embedded in LR White.
Ultrathin sections (80 nm), which were prepared with an ultramicrotome equipped with a diamond knife and mounted on uncoated nickel grids (300 mesh), were stained with 4% uranyl acetate and 1% lead citrate. The sections were observed with a JEM-1400 TEM (JEOL Ltd.) at an acceleration voltage of 80 kV. Cell wall thickness was calculated with the iTEM image analysis program (Olympus Soft Imaging Solution GmbH).

Neutral sugar analysis by gas chromatography
The neutral sugar contents were analyzed using a modified alditol acetate method [43].
The cell wall, isolated with acetone and distilled water, was treated with 72% sulfuric acid at RT for 45 min and diluted with water to 4% sulfuric acid. After hydrolysis for 1 h at 121 °C, the solution was neutralized with ammonia solution. After myo-inositol was added as an internal standard, an aliquot was reduced using 2% sodium tetrahydroborate. Alditol was acetylated with a catalyst (methylimidazole) followed by acetic anhydride, and it was then extracted with dichloromethane. Gas chromatography (CP-9100, Chrompack, the Netherlands) was performed using a DB-225 capillary column (30 m x 0.25 mm i.d., 0.25-μm film thickness, J&W) and a flame ionization detector. The operating conditions were as follows: detector temperature, 250 °C; injector temperature, 220 °C; oven temperature rose from 100 (1.5 min) to 220 °C at a rate of 5 °C•min -1 . Compounds were identified by comparing retention times of standard compounds.

Quantitative real-time PCR (qRT-PCR) analysis
Purified total RNA (1 μg) was used for first-strand cDNA synthesis with oligo d(T)18 and reverse transcriptase according to the manufacturer's instructions (PhileKorea Technology, Korea) [28]. Experimental samples were evaluated in triplicate, and qRT-PCR reactions for each sample were run in duplicate. PCR was conducted using a LightCycler ® 480 Ⅱ Real  [44], and as an internal control, qRL25 was selected for data normalization. The nucleotide sequences of other primers are listed in Table   SI.

Microarray analysis
Expression profiling was conducted using a tobacco 3'-Tiling microarray containing 34,052 consensus sequences that were deposited into the National Center for Biotechnology Information database (NCBI; http://www.ncbi.nlm.nih.gov/). The microarray data sets used in this study can be found at www.ncbi.nlm.nih.gov/geo/ (Gene Expression Omnibus, GEO). The NCBI accession number is GSE47671 [27].

RT-PCR analysis
The frozen cells were ground in liquid nitrogen and kept frozen. Total RNA was extracted from cultured calli or plants using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) [27,28]. First-strand cDNA synthesis was performed with 2 μg of total RNA and an oligo (dT) primer using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer's recommendations. For the semiquantitative PCR analysis, Taq polymerase was used.
Amplification was performed using cycles of 95°C for 15 s, 57°C for 20 s, and 72°C for 30 s.
The PCR products were electrophoresed on 1.2% agarose gels in the presence of ethidium bromide (0.5 mg·L −1 ). Each PCR was performed at least 3 times with different cDNA sets and primers (Table S1).

Consent for publication
Not applicable.

Availability of data and materials
Datasets used in the current study are available from the corresponding author on reasonable request.          Table S1.
Supplementary Figure 3. Expressions of SAM synthase, which produce S-adenosylmethione (SAM), a precursor for ethylene. Their expressions are slightly induced. Each sample was harvested from 3-days old culture. Error bars represent standard error from the mean (n=3).