Membrane trafficking mediated by OsDRP2B is specific for cellulose biosynthesis

Abstract

Increasing evidence has revealed that membrane trafficking is highly associated with cell wall metabolism. Factors involved in vesicle delivery, e.g., cytoskeleton and motor proteins, have showed regulatory effects on cell wall structure and components. However, little is known about the involvement of other trafficking components in distribution of cell wall-related compartments. Dynamins are important proteins functioning in membrane tubulation and vesiculation. Recently, we have reported characterization of the rice dynamin-related protein 2B (OsDRP2B). Mutation in OsDRP2B causes a significant reduction in cellulose content. Its association with the trans-Golgi network (TGN) and clathrin-coated vesicles and the reduced CESA4 abundance at the bc3 plasma membrane suggest that BC3/OsDRP2B is involved in the transport of essential elements for cellulose synthesis. Here, we provide additional evidence for BC3 subcellular localization via observing OsDRP2B-GFP in living root hairs of transgenic plants. Uronic acid and fractional composition analyses further confirm that the amount of arabinoxylan and other noncellulosic polysaccharides is increased in bc3. However, three putative xylan synthesis genes are downregulated in mutant plant revealed by real-time PCR analysis. These results imply that compartments delivered by OsDRP2B are specifically responsible for cellulose biosynthesis.

Plant cell wall is an extracellular matrix enriched in polysaccharides. Except for cellulose that is produced at the plasma membrane by cellulose synthase (CESA) complexes, most of the cell wall products are assumed being synthesized inside cells, e.g., in the Golgi apparatus and secreted outside through complex membrane trafficking. Besides the cell wall-localized products, some proteins essential for cellulose biosynthesis need to be translocated onto the plasma membrane to facilitate cellulose formation.^1,2 Intracellular trafficking is therefore a key level for regulating cell wall composition and architecture, which are highly dynamic during cellular development.³ This notion is substantiated by the fact that wall architecture within the same cell is heterogeneity, indicating the presence of cell wall specific deposition domains.^4,5 For example, pectins are often located at the cell corners.³ Different de-esterified homogalacturonan (HG) are present along the growing pollen tubes or root hairs: tips have highly esterified HG; the de-esterified degree is increased after tips.⁶ Although it is believed that these specific patterns could be the result of the targeted secretion of polysaccharides,³ our knowledge about the polysaccharide secretion is still very few. Currently, in vivo viewing CESA-containing compartments and the movement inside living cells have provided direct evidence for the trafficking action of CESA compartments.^2,7,8 The delivery and removal of CESA complexes to/from the plasma membrane are very complicated, which require the involvement of many components, such as cytoskeleton and syntaxins.^7,9,10 Syntaxins, part of SNARE complexes, function as docking factor of cell wall-related compartments during cell plate formation.¹⁰ Dynamin and dynamin-related proteins (DRPs) are involved in diverse events of cellular membrane remodeling.¹¹ It remains unknown about whether DRPs are responsible for CESA trafficking. Recently, we have reported that BC3, the rice DRP2B protein, plays a role in complex membrane trafficking and affects the biosynthesis of secondary walls. Here, we provide additional cellular and wall chemical data to confirm that BC3/OsDRP2B is specifically involved in the secondary cell wall cellulose synthesis.

BC3/OsDRP2B is Involved in Membrane Trafficking

We have identified that BC3/OsDRP2B is targeted either to the trans-Golgi network (TGN) and clathrin-coated vesicles (CCV) or to the endosomes labeled by FM4-64.¹² BC3/OsDRP2B is therefore very likely involved in endocytosis and/or post-Golgi trafficking. Root hair is a kind of typical tip-growing cells, in which the endocytosis and extocystosis are extremely activated because proteins and polysaccharides required for the tip-growth need to be translocated or recycled through membrane trafficking.^13,14 To examine whether BC3/OsDRP2B participates in root hair growth, we in vivo detected the GFP signals in root hairs of transgenic plants overexpressing OsDRP2B-GFP. The signals of BC3/OsDRP2B were present as punctuate, reminiscent of some activated compartments (Fig. 1A and B). We also found that BC3/OsDRP2B is expressed in root hairs based on the GUS activity detected in the BC3_pro::GUS transgenic plants (Fig. 1C and D). BC3/OsDRP2B indeed functions in root hairs. To address what function BC3/OsDRP2B performs there, we compared the length of root hair between bc3 and wild-type plants. Unexpectedly, bc3 has longer root hairs than the wild type (data not shown). Therefore, BC3/OsDRP2B is involved in membrane trafficking in root hair cells, where it may play a negative role for their elongation.

Figure 1.

Localization of OsDRP2B-GFP and OsDRP2B expression in root hair. (A and B) OsDRP2B-GFP signals in root hairs (A) and its DIC image (B). (C and D) GUS activity in root hairs of BC3pro::GUS transgenic plants (C) and its enlargement (D). Bars = 3 µm.

bc3 Mutation Causes Increased Level of Several Cell Wall Components

We have reported that bc3 mutation decreases the cellulose content and increases many matrix polysaccharides.¹² To figure out whether it affects the synthesis of pectic polysaccharides, we examined uronic acid content through analyzing tetramethylsilane derivatives of culm residues. Galacturonic acid (GalUA) and glucuronic acid (GlcUA) were significantly increased in bc3 mutant plants (Table 1). GalUA is an important sugar presenting in HG, rhamnogalacturonan I and rhamnogalacturonan II. Therefore, mutation in BC3/OsDRP2B causes the increase of pectin amount. In addition, sugars representing matrix polymers were also generally increased (Table 1). To assign the alterations to specific wall polymers, we fractionated the culm residues by sequential chemical/enzyme extractions (endopolygalaturonase and EGTA-Na₂CO₃ treatments for pectin extraction and 1 N and 4 N potassium hydroxide (KOH) treatments for hemicellulose extraction). Remarkably, increases in galactose, glucose, arabinose and xylose were detected in 1 N and 4 N KOH fractions (Table 2). Xylose and arabinose are two sugars of arabinoxylan, a major hemicellulose in rice. However, the ratio of arabnose and xylose is not significantly changed in bc3. Above results suggest that disruption of BC3/OsDRP2B increases the amount of arabinoxylan without altering its structure.

Table 1.

Monosaccharide compositional analysis of wall residues from mature culms of the wild-type and bc3 plants

Samples	Rhamnose	Fucose	Arabinose	Xylose	Mannose	Galactose	Glucose	GalUA	GlcUA
Wt	2.5 ± 0.1*	0.6 ± 0.0	72.1 ± 3.3*	280.6 ± 6.7*	0.4 ± 0.0	7.4 ± 0.1*	30.3 ± 0.6	7.7 ± 0.4*	3.9 ± 0.2*
bc3	3.5 ± 0.2*	0.8 ± 0.1	97.7 ± 5.1*	362.8 ± 16.8*	0.5 ± 0.0	11.9 ± 0.3*	41.7 ± 10.7	9.4 ± 0.3*	5.4 ± 0.3*

Alcohol-insoluble residues (AIR) were prepared from the 2^nd internodes of bc3 and wild type. The tetramethylsilane derivatives were analyzed by GC-MS for glycosyl residue composition. The results were given as means (mg/g of AIR) of three independent assays ± SD.

^*Significance between the wild type and mutant is determined by the least significant difference t test at p < 0.05. GalUA, galacturonic acid; GlcUA, glucuronic acid.

Table 2.

Monosaccharide compositional analysis of wall fractions prepared from mature culms of wild-type and bc3 plants

Fractions	Samples	Rhamnose	Fucose	Arabinose	Xylose	Mannose	Galactose	Glucose
EPG	Wt	0.3 ± 0.0	ND	1.9 ± 0.2	4.0 ± 0.6	3.4 ± 0.8	2.2 ± 0.2	28.8 ± 3.4
	bc3	0.4 ± 0.1	ND	1.6 ± 0.3	2.6 ± 0.2	5.3 ± 0.5	2.5 ± 0.3	26.4 ± 3.1
Na2CO3	WT	ND	ND	0.3 ± 0.1	0.3 ± 0.0	0.3 ± 0.1	0.3 ± 0.1	1.3 ± 0.1
	bc3	ND	ND	0.3 ± 0.2	0.4 ± 0.1	0.2 ± 0.0	0.5 ± 0.1	1.0 ± 0.1
1 N KOH	WT	0.4 ± 0.2	ND	22.5 ± 2.1*	107.7 ± 9.3*	0.2 ± 0.1	5.6 ± 0.8*	13.7 ± 2.1*
	bc3	0.4 ± 0.2	ND	35.8 ± 1.7*	148.4 ± 13.7*	0.3 ± 0.1	10.3 ± 1.6*	20.7 ± 0.6*
4 N KOH	WT	0.4 ± 0.2	ND	6.7 ± 1.1*	18.6 ± 2.1*	0.1 ± 0.0	6.6 ± 0.9*	10.7 ± 1.8*
	bc3	0.6 ± 0.2	ND	11.9 ± 0.9*	35.4 ± 3.3*	0.1 ± 0.0	11.7 ± 0.6*	16.3 ± 1.4*

Wall fractions were prepared from the 2^nd internodes' AIRS of bc3 and wild type. After starch being removed, the AIR was sequentially fractionated by endopolygalaturonase (EPG) and EGTA-Na₂CO₃ (5 mm EGTA and 50 mm Na₂CO₃) followed by 1 N and 4 N potassium hydroxide (KOH). Soluble components were neutralized and dialyzed against water. The amount of sugar residues was determined by GC-MS analysis of alditol acetate derivatives. Data were means (mg/g AIR) of four independent assays ± SD. ND, none detected.

^*Significance between the wild type and mutant is determined by the least significant difference t test at p < 0.05.

It has been revealed that several Arabidopsis glycosyltransferases are involved in xylan synthesis: IRX7 (Irregular Xylem7), IRX8 (Irregular Xylem8) and PARVUS are responsible for the primer formation; IRX9 (Irregular Xylem9) and IRX14 (Irregular Xylem14) participate in xylan backbone synthesis.^15–18 Considering that the walls of bc3 have an increased level of xylan, we analyzed the expression level of three homologous genes putative for xylan synthesis (IRX7L, IRX14L and PARVUSL) in bc3 and wild-type. As shown in Figure 2, the three genes are downregulated in the mutant. The inconsistent alteration between xylan amount and gene expression level indicates that raised xylan content might be indirect or feedback effects of bc3 mutation. This result further confirmed that less cellulose amount may be a direct effect of BC3 disruption. Production of cell wall polysaccharide is highly co-regulated in high plants.¹⁹ Blocking cellulose synthesis often results in the increase of non-cellulose polysaccharides. Cellulose deficient mutant bc11 (OsCESA4 mutant) in rice and mur10 (AtCESA7 mutant) in Arabidopsis have increased level of xylan and xyloglucan, respectively.^20,21 On the contrary, deficiency in the synthesis of polysaccharide matrix or glycoprotein impedes the cellulose synthesis, probably through aberrant assembly of nascent cellulose microfibrils at the plasma membrane. Both Arabidopsis cyt1 mutant that is defecient in mannose-1-phosphate guanylyltransferase for N-glycosylated protein formation and the rice bc10 mutant that disturbs the production of a certain glycoprotein or matrix polysaccharide cause reduced cellulose accumulation.^22,23 Therefore, co-regulatory machinery for cell wall biosynthesis may be common in dicot and monocot plants.

Figure 2.

Expression level of putative xylan synthesis genes in wild type and bc3. Real-time PCR analysis of IRX7L, IRX14L and PARVUSL expression. The expression levels in wild type are considered as 1.

Conclusions

Our data indicate that BC3/OsDRP2B participates in the endocytic pathway probably as well as in post-Golgi membrane trafficking.¹² Increased noncellulosic polysaccharide content in bc3 also implies that vesicle trafficking responsible for transporting or recycling noncellulosics is not blocked in bc3, reminiscent of Arabidopsis DRP1A null mutant, in which several noncellulosics levels are increased.²⁴ The raised matrix polysaccharides may result from the normal function of other DRP2 or DRP members, which sustain the transport of these components. Therefore, vesicle trafficking mediated by BC3/OsDRP2B seems specific for cellulose synthesis. Further studies need to be performed for doubtlessly understanding the function of BC3/OsDRP2B in rice plants.

Addendum to: Xiong G, Li R, Qian Q, Song X, Liu X, et al. The rice dynamin-related protein DRP2B mediates membrane trafficking and thereby plays a critical role in secondary cell wall cellulose biosynthesis. Plant J. 2010;64:56–70. doi: 10.1111/j.1365-313X.2010.04308.x.