Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Expression vector wikipedia , lookup
Biochemical cascade wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Paracrine signalling wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Signal transduction wikipedia , lookup
Plant Cell Physiol. 49(10): 1522–1535 (2008) doi:10.1093/pcp/pcn120, available online at www.pcp.oxfordjournals.org ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] The Glycerophosphoryl Diester Phosphodiesterase-Like Proteins SHV3 and its Homologs Play Important Roles in Cell Wall Organization Shimpei Hayashi 1, 2, Tadashi Ishii 3, Toshiro Matsunaga 4, Rumi Tominaga 2, Takashi Kuromori 2, Takuji Wada 2, Kazuo Shinozaki 2 and Takashi Hirayama 1, 5, * 1 International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama, 230-0045 Japan RIKEN Plant Science Center, 1-7-22 Tsurumi, Yokohama, 230-0045 Japan 3 Forestry and Forest Products Research Institute, 1 Matunosato, Tsukuba, Ibaraki, 305-8687 Japan 4 National Agricultural Research Center, National Agriculture and Food Research Organization, 3-1-1 Kannondai, Tsukuba, Ibaraki, 305-8666 Japan 5 RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan 2 Despite the importance of extracellular events in cell wall organization and biogenesis, the mechanisms and related factors are largely unknown. We isolated an allele of the shaven3 (shv3) mutant of Arabidopsis thaliana, which exhibits ruptured root hair cells during tip growth. SHV3 encodes a novel protein with two tandemly repeated glycerophosphoryl diester phosphodiesterase-like domains and a glycosylphosphatidylinositol anchor, and several of its paralogs are found in Arabidopsis. Here, we report the detailed characterization of mutants of SHV3 and one of its paralogs, SVL1. The shv3 and svl1 double mutant exhibited additional defects, including swollen guard cells, aberrant expansion of the hypocotyl epidermis and ectopic lignin deposits, suggesting decreased rigidity of the cell wall. Fourier-transform infrared spectroscopy and measurement of the cell wall components indicated an altered cellulose content and pectin modification with cross-linking in the double mutant. Furthermore, we found that the ruptured root hair phenotype of shv3 was suppressed by increasing the amount of borate, which is supposed to be involved in pectic polysaccharide cross-linking, in the medium. These findings indicate that SHV3 and its paralogs are novel important factors involved in primary cell wall organization. Keywords: Arabidopsis thaliana — Cell wall — Cellulose — GPI-anchored protein — Pectin — Root hair. Abbreviations: AIR, alcohol-insoluble residue; AVG, 1-aminoethoxyvinyl glycine; CaMV, cauliflower mosaic virus; FTIR, Fourier-transform infrared; GFP, green fluorescent protein; GPD, glycerophosphoryl diester phosphodiesterase; GPDL, GPD-like; GPI, glycosylphosphatidylinositol; GUS, b-glucuronidase; PC, principal component; RG-II, rhamnogalacturonan-II; RT–PCR, reverse transcription–PCR; SHV3, SHAVEN3; SVL, SHV3-like. Introduction Plant shape and size are determined primarily by cell division and expansion. Cell expansion involves controlled cell wall organization. Primary walls require both mechanical stability and extensibility to allow for cell expansion without cell rupture due to excessive turgor pressure. Cell walls must be able selectively to loosen, integrate newly synthesized cell wall components, and form a new cell wall matrix quickly and correctly during cell expansion (Cosgrove 2005). Therefore, cell expansion requires the organized disruption and formation of linkages among cell wall components. Because of the complex structure of cell walls, understanding the regulatory mechanisms of cell wall organization during cell expansion and shape change is a major challenge in plant science. Primary cell walls, which are deposited during cell expansion, are composed mainly of cellulose microfibrils, hemicelluloses, pectins and proteins. The cellulose microfibrils, which are crystallized fibers of b-1,4-glucose chains, are synthesized by the cellulose synthase complex at the plasma membrane. The other components, including the hemicelluloses and pectins, are synthesized in the Golgi apparatus then secreted into the cell wall matrix (Scheible and Pauly 2004, Cosgrove 2005). Cell walls have a complex structure comprising several different types of linkages between their components. Hemicelluloses, such as xyloglucan, form hydrogen bonds with the surfaces of cellulose microfibrils and other polysaccharides. One of the pectic components, rhamnogalacturonan II, forms borate diester cross-links between apiose residues (O’Neill et al. 2004). Another pectic component, homogalacturonan, forms calcium cross-links between carboxyl groups in a methyl esterification-dependent manner (Ridley et al. 2001). Understanding cell wall organization requires identifying not only the components but also the factors that modulate the components and comprise the network *Corresponding author: E-mail, [email protected]; Fax, þ81-45-508-7363. 1522 SHV3 and SVLs in cell wall organization structure of the cell wall. Several enzymes that modify the linkages between the components of the cell wall have been identified. Transglycosylation between free and cellulosebound xyloglucans catalyzed by xyloglucan endotransglycosylase is thought to be involved in cell wall loosening (Vissenberg et al. 2003). Pectin methylesterases, which remove the methyl groups of pectins in the cell wall, control the number of calcium cross-links among the pectins. The vgd1 mutant, an Arabidopsis line defective in pectin methylesterase, exhibits slow-growing pollen tubes leading to reduced male fertility in vivo and unstable pollen tubes that are susceptible to bursting in vitro (Jiang et al. 2005). Expansins are thought to disrupt the non-covalent interactions between cell wall polymers in acid-induced cell growth, although their molecular functions have not yet been elucidated (Li et al. 2003). In addition, recent studies have revealed that a large number of proteins predicted to be located in the cell wall have important roles in cell wall organization. Several glycosylphosphatidylinositol (GPI)anchored proteins that attach to the outer cell surface have been functionally characterized in cell wall organization. COBRA is required for the deposition of cellulose and the regulation of anisotropic expansion, and cobra mutants have swollen roots because of abnormal radial cell expansion (Schindelman et al. 2001, Roudier et al. 2005). SKU5 is structurally similar to multiple copper oxidases, and its defective mutant exhibits an altered root waving pattern (Sedbrook et al. 2002). A mutant of SOS5, which encodes a protein with fasciclin-like and arabinogalactan protein-like domains, exhibits arrested root growth and root swelling under salt stress conditions (Shi et al. 2003). It has been postulated that these factors are involved in the construction of cell wall networks. Root hair cells exhibit tip growth and a fast rate of expansion. A sophisticated regulatory mechanism is believed to maintain the organization of the cell wall to prevent the tip from rupturing during root hair elongation. Ruptured root hairs have been observed only in the kojak (kjk) (Favery et al. 2001), lrx1lrx2 (Baumberger et al. 2003) and mrh4 (Jones et al. 2006) mutants, which are thought to have abnormal cell wall organization. To learn more about the mechanism of cell wall organization, we isolated shaven3 (shv3) from a screen of Ds insertional mutants for lines with abnormally shaped root hairs. SHV3 encodes a GPIanchored protein similar to glycerophosphoryl diester phosphodiesterase (GPD) and is required for root hair elongation (Parker et al. 2000, Jones et al. 2006). In this study, we found that a combination of shv3 and a disruptant mutation in a paralog conferred additional unique phenotypes to the morphology of the epidermal cells and a number of phenotypes common to cell wall-related mutants. Furthermore, an analysis of the cell wall components in the double mutant revealed an altered 1523 crystalline cellulose content with pectin modifications and changes in the amount of cross-linking. These findings suggest that SHV3 and its paralogs encode novel proteins with important roles in cell wall organization. Results Identification of the SHV3 locus by Ds transposon tagging We isolated a recessive mutant defective in root hair formation from a screen of RIKEN Arabidopsis Ds transposon insertion lines (Kuromori et al. 2004, Kuromori et al. 2006). In the mutant line 15-1096-1, tip growth in nearly all root hair cells was blocked due to a rupture at the tip (Fig. 1A). However, bulges were observed at the appropriate positions in the trichoblasts, indicating normal root hair development. No other visible phenotypes were observed in this mutant. The transposon element was found to be inserted into At4g26690, which encodes a GPD-like protein (Fig. 1C) that is predicted to have a glycosylphosphatidylinositol (GPI) anchor sensitive to phosphatidylinositol-specific phospholipase C (Borner et al. 2003). This gene was recently identified as the gene responsible for the root hair-defective mutant, shv3/mrh5 (Parker et al. 2000, Jones et al. 2006). Jones et al. (2006) found that the expression of At4g26690 was correlated with the root hair morphogenesis, and T-DNA insertion mutations of this gene caused a root hair-defective phenotype. All of the F1 plants generated by crossing 15-1096-1 with shv3 had the same defective root hair phenotype as the parents, indicating that this Ds mutation is allelic to shv3 (Fig. 1B). We examined the nucleotide sequence of this gene in the shv3 mutant and identified a C-to-T transversion mutation causing an amino acid substitution Thr(364) to Ile, consistent with the effect of ethylmethanesulfonate, which was used for mutagenesis (data not shown) (Parker et al. 2000). We obtained a T-DNA insertion mutant (SALK_024208) of this gene and confirmed that it conferred the same phenotype. In the previous study, SALK_024208 was named mrh5-3 (Jones et al. 2006). To avoid confusion, in this study, we refer to the mutant isolated by Parker et al. (2000) as shv3-1, the T-DNA insertion line as shv3-2, and the Ds insertion line 15-1096-1 as shv3-3; shv3-2 was used in the following experiments. In shv3-2 and shv3-3, transcript fragments were detected by reverse transcription–PCR (RT–PCR) (Supplementary Fig. S1). We assumed that the shv3-2 and shv3-3 products were not functional, however, because the predicted polypeptides lack the N-terminal signal sequence or the C-terminal region required for correct protein localization (see below). Localization of SHV3 The predicted SHV3 protein contains a putative signal sequence at the N-terminus and a putative GPI anchor 1524 SHV3 and SVLs in cell wall organization A B Ds15-1096-1 x shv3-1 F1 Ds15-1096-1 x WT F1 C shv3-3 Ds15-1096-1 shv 3-1 C>T shv3-2 SALK024208 TGA 2280 ATG 1 GPD-like GPD-like AGP GPD-like AGP 759aa signal sequence GPD-like GPI-anchore D Fig. 1 Identification of the shv3 mutant allele and SHV3 localization. (A) Phenotype of Ds15-01096-1. The primary root (left and middle) and root hair (right) of 4-day-old Ds5 (control) and Ds15-1096-1 seedlings. The root hair is ruptured and tip growth is blocked in Ds15-1096-1. (B) Allelism between Ds15-1096-1 and shv3 (Parker et al. 2000). The F1 progeny of a test cross between Ds15-1096-1 and shv3 exhibited the parental phenotype. (C) Structure of the SHV3 gene and its translation product. addition site (omega site) near the C-terminus, indicating its localization at the plasma membrane (Borner et al. 2002). To confirm this assumption, we generated transgenic plants expressing a chimeric green fluorescence protein (GFP)– SHV3 protein with GFP attached to the C-terminal end of the SHV3 signal sequence. The root hair phenotype of shv3 was completely restored by transformation with this construct (data not shown), indicating that the fusion protein was fully functional. GFP fluorescence was observed at the plasma membrane (Fig. 1D, upper panels), suggesting its function at the cell surface. In addition to the plasma membrane, GFP fluorescence was often observed in some intracellular structures (Fig. 1D, lower panels and Supplementary Fig. S2). This intracellular fluorescence may be derived from the GPI-anchored SHV3 protein on the membrane trafficking pathway. In the root hair cell, the fluorescence was not polarized at the tip where SHV3 is supposed to function and was observed in a wide area of cell surface and intracellular structures. The fluorescence intensity varied among the cells but was low in most cases, despite the fact that expression of the transgene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. Exceptionally strong and stable fluorescence was observed in the trichomes; however, little fluorescence was detected at the basal surface of the trichomes, indicating the polarized localization of SHV3 (Fig. 1D and Supplementary Fig. S2). Glycerophosphoryldiester phosphodiesterase-like genes SHV3 has two tandemly repeated GPD-like domains, though the biochemical function of these domains is unknown (Fig. 2A). Using the MIPS database (http:// mips.gsf.de/), we identified six genes from Arabidopsis that are closely related to SHV3 [GPDL1 (glycerophosphodiesterase-like) to GPDL6]. Other genes encoding GPD-like proteins exist in the Arabidopsis genome, however. Thus, to avoid confusion, in this study we refer to GPDL1 (At5g55480), 3 (At1g66970), 4 (At3g20520), 5 (At5g58050) and 6 (At5g58170) as SVL (SHV3-like) 1, 2, 3, 4 and 5, respectively (Supplementary Table S1). A BLAST search identified putative orthologs of SHV3 only in plants (e.g. rice). A transcript encoding a polypeptide with high similarity to SHV3 was also found in Physcomitrella patens (Physcobase, http://moss.nibb.ac.jp). In addition to SHV3 (At4g26690) encodes a protein containing tandemly repeated glycerophosphoryl diester phosphodiesterase (GPD)-like domains and an arabinogalactan protein (AGP)-like domain. The mature protein is presumably modified with a glycosylphosphatidylinositol (GPI) anchor. (D) Confocal imaging of cells expressing the GFP–SHV3 fusion protein. GFP fluorescence (green) and chlorophyll autofluorescence (red) in the hypocotyl (upper and lower left), and a root hair (lower right) of a transgenic plant expressing GFP::SHV3 under control of the CaMV 35S promoter is shown. An arrowhead indicates intracellular structures. Bar ¼ 20 mm. SHV3 and SVLs in cell wall organization A SHV3 & SVLs GPD-like N 1525 GPD-like C At1g74210 C E. coli UgpQ At5g08030 E. coli GlpQ E. coli GlpQ B shv3-1, T>I SVL5-C SVL4-C SVL2-C At1g74210 At5g08030 SHV3-C SVL1-C SVL3-N SVL3-C SVL1-N SVL5-N SVL4-N SVL2-N SHV3-N 0.1 Fig. 2 Comparison of the primary sequences of SHV3, SVLs and GPDs. (A) Schematic representation of SHV3, SVLs, two Arabidopsis candidate GPDs (At1g74210 and At5g08030) and an E. coli GPD (GlpQ). The gray portions indicate the most conserved parts of the GPDlike domains (i.e. the residues that were used for alignment in B). (B) Alignment of the residues from the most conserved parts of the GPDlike domains and GPDs. Those residues thought to interact with the substrate or calcium ions are indicated by open and filled triangles, respectively. The shv3-1 transversion was observed in the C-terminal GPD-like domain. Clustal X (Thompson et al. 1997) was used to create the alignment. Amino acids identical in all sequences are highlighted in black, while those identical in at least eight sequences are highlighted in gray. (C) Phylogenic tree of the GPD-like domains and GPDs. The phylogenic distances were calculated by comparing the primary sequences of the GPD-like domains and GDPs using Clustal X. The phylogenic tree was drawn using TreeView (Page 1996). The bar indicates 0.1 substitutions per site. SHV3 and SVL genes, a gene (At1g66980) encoding a protein with a similar domain organization and an additional Ser/Thr kinase domain was found in Arabidopsis. Because this gene has 480% identity to the flanking gene (SVL2), and as no other similar gene was found in any other plant species, it may be an Arabidopsisspecific gene produced by chromosomal rearrangement. We examined a disruptant mutant of At1g66980 (Ds 12-1469-1), but failed to detect any visible phenotype (data not shown). GPD catalyzes the hydrolysis of glycerophosphoryl diester to a glycerol 3-phosphate and alcohol. Carrot GPD activity was detected in cell wall fractions (Van Der Rest et al. 2004). In Arabidopsis, At1g74210 and At5g08030 are thought to be equivalent to the carrot GPD, based on their peptide sequences. To compare the GPD-like domains of SHV3 and SVLs, their primary sequences were aligned with those of the putative Arabidopsis GPDs and a known GPD in Escherichia coli, GlpQ (Larson et al. 1983, Tommassen et al. 1991) (Fig. 2). The crystal structure of E. coli GlpQ and glycerol complex (PDB: 1ydy) revealed a number of residues that are probably essential for interactions with the substrate and calcium ions. These residues are conserved in At1g74210 and At5g08030, but not in SHV3 or SVLs, suggesting that the biochemical function of the GPD-like domains in SHV3 and SVLs is distinct from that in typical GPD (Fig. 2). A comparison of the N- and C-terminal GPD-like domains indicated that these two domains have distinct sequences, suggesting that they have different biochemical functions. The recessive phenotype caused by the shv3-1 missense mutation in the C-terminal GPD-like domain indicates that this domain is necessary for SHV3 function. We attempted to determine whether recombinant SHV3 produced in E. coli has GPD activity using glycerophosphocholine as a substrate, but we were unable to detect any such activity (Supplementary Fig. S6). Overlapping and distinct distributions of the expression of SHV3 and SVLs in tissues To determine the tissue-specific expression pattern of SHV3 and SVL genes, RT–PCR analysis was performed using total RNA isolated from the aerial parts or roots of 7-d-old seedlings, and dissected tissues from 6-week-old plants (Fig. 3). In 7-d-old seedlings, only SHV3, SVL1 and SVL2 transcripts were detected. SHV3 and SVL1 mRNA was detected in both the aerial parts and roots, whereas SVL2 mRNA was detected only in the aerial parts. SHV3, SVL1 and SVL2 mRNA was detected in various 6-week-old vegetative tissues. SVL3 mRNA was detected in the siliques 1526 SHV3 and SVLs in cell wall organization 7-day-old 6-week-old Aerial Root Rosette Cauline Root Leaf Leaf Stem Flower Silique SHV3 SVL1 SVL2 SVL3 A number of intriguing novel phenotypes were observed in shv3-2svl1-1, but not in shv3-2svl2-1 or svl1-1svl2-1. Additionally, we generated an shv3-2svl1-1svl2-1 triple mutant, but its phenotype did not differ from those of the double mutants. We also tried unsuccessfully to generate an svl4svl5 double mutant presumably due to small genetic distance between them. SVL4 SVL5 rRNA Fig. 3 RT–PCR analysis of the expression SHV3 and SVL genes. The expression of SHV3 and SVL genes in 7-d-old seedlings and 6-week-old plants was analyzed by semi-quantitative RT–PCR using gene-specific primers. 18S rRNA was used as an internal control. and, to a lesser extent, flowers. SVL4 and SVL5 mRNA was detected predominantly in the flowers, consistent with data showing that SVL4 and SVL5 are expressed specifically in pollen (Lalanne et al. 2004). To examine the detailed expression patterns of SHV3, SVL1 and SVL2, we performed a histochemical analysis of transgenic plants harboring the b-glucuronidase (GUS) gene fused to the putative promoter regions of each of these genes (Fig. 4). In the PSHV3::GUS transgenic plants, GUS activity was strongest in the root hair cells (Fig. 4A, J, K). The appearance of GUS activity in the root hair cells appeared to coincide with the initiation of root hair formation (Fig. 4J), consistent with previous data (Jones et al. 2006). GUS activity was also detected in the petioles, hypocotyls (Fig. 4L) and young leaves (Fig. 4D, M). PSVL1::GUS activity was strongest in the vascular tissues (Fig. 4B, E, P, Q, T) and root meristems (Fig. 4B, N, O). Additionally, GUS activity was observed in the hypocotyl epidermis (Fig. 4P), young leaf guard cells (Fig. 4S) and trichomes (Fig. 4R). PSVL2::GUS activity was strongest in the leaves (Fig. 4C, F, V). GUS activity was also detected in the hypocotyls of dark-grown PSHV3::GUS and PSVL1::GUS transgenic plants (Fig. 4G, H). GUS activity in PSVL2::GUS was detected in light-grown hypocotyls (Fig. 4U), but not in dark-grown hypocotyls (Fig. 4I). These results demonstrate that the expression of SHV3 and SVL genes is strictly regulated and that they have overlapping and distinct tissue expression patterns. Characterization of the SVL disruptant mutants To investigate whether the disruption of SVL genes would produce an shv3-like phenotype, we screened SVL T-DNA insertion lines (Fig. 5A). Presumably because of functional redundancy among these gene products, no visible phenotype was observed. Therefore, we generated mutants of SHV3, SVL1 and SVL2 because our earlier data indicated that these genes are expressed in various tissues. The shv3-2svl1-1 double mutant exhibits altered cell wall organization and cell expansion The shv3-2svl1-1 double mutant seedlings exhibited increased anthocyanin accumulation (data not shown) and frequently collapsed trichomes (Supplementary Fig. S3). In addition, some brown discoloration was observed around the endodermis in the hypocotyl and root (Fig. 5B). This phenotype was initially observed in the lower part of the hypocotyl then gradually spread to the upper hypocotyl and root (data not shown). Histochemical analysis using phloroglucinol-HCl revealed ectopic lignification at the discolored sites in the double mutant, but not in any of the monogenic mutants (Fig. 5B). Ectopic lignification was previously observed in several mutants showing altered cell wall organization and cell expansion regulation with shortened dark-grown hypocotyls, including eli1/cev1 (Cano-Delgado et al. 2000, Ellis et al. 2002), kor1 (Nicol et al. 1998), elp1/ctl1 (Zhong et al. 2002) and kob1/abi8 (Pagant et al. 2002, Brocard-Gifford et al. 2004). Therefore, we investigated whether the shv3-2svl1-1 double mutant had a similar phenotype. Four-day-old dark-grown shv3-2svl1-1 seedlings displayed shorter and thicker hypocotyls than the wild type (Fig. 5C, D), which is consistent with the hypocotyl expression of SHV3 and SVL1 as indicated by GUS staining in the promoter–GUS transgenic lines (Fig. 4P, Q). The eli1/cev1 mutant, which is defective in a cellulose synthase subunit (CESA3), overproduces ethylene and jasmonic acid, and accumulates transcripts of their responsive genes. Similar phenotypes have been reported in the elp1/ctl1 and kob1/abi8 mutants, and it has been proposed that ethylene and jasmonic acid signaling is activated by alterations in cell wall organization. To determine whether such signaling is activated in shv3-2svl1-1, the transcript levels of PDF1.2 and VSP1 were examined by RT–PCR using total RNA extracted from 7-d-old plants. PDF1.2 expression is induced by jasmonic acid and ethylene (Penninckx et al. 1998), whereas VSP1 is induced by jasmonic acid but not ethylene (Rojo et al. 1999). Strong mRNA expression of PDF1.2 and VSP1 was detected in the double mutant, whereas that in the wild type was relatively weak (Fig. 5E). The shortened dark-grown hypocotyls of eli1/cev1 and elp1/ctl1 are restored by etr1, a strong ethylene-insensitive mutation, and partially by inhibitors of ethylene production [e.g. 1-aminoethoxyvinyl glycine SHV3 and SVLs in cell wall organization A B C J K L O P Q T U 1527 D E F G H I N M R S V Fig. 4 Promoter activity of SHV3, SVL1 and SVL2. Histochemical analysis of transgenic plants harboring the GUS gene under control of the putative SHV3 (A, D, G, J–M), SVL1 (B, E, H, N–T) and SVL2 (C, F, I, U, V) promoters. (A–C) Seven-day-old seedlings. (D–F) Aerial parts of 3-week-old seedlings. (G–I) Four-day-old dark-grown seedlings. (J) Root of a 7-d-old seedling. (K) Root cross-section. Arrowheads indicate the root hair cells. (L) Upper portion of the hypocotyl from an 8-d-old seedling. Arrowheads indicate the petioles. (M) Leaf at an early developmental stage (indicated by a broken line). (N) Root tip. (O) Lateral root primordium (arrowhead). (P) Lower hypocotyl and root. (Q) Root cross-section. (R) Leaves at an early developmental stage. (S) Guard cells. (T) Leaf cross-section. Arrowheads indicate the vascular tissues. (U) Hypocotyl of a 3-d-old seedling. (V) Leaf cross-section. (AVG)] or binding (Agþ), respectively. Unlike the elp1/ctl1 mutants, AVG did not restore the shortened dark-grown hypocotyls of shv3-2svl1-1 (Supplementary Fig. S4), which suggests that the shv3-2svl1-1 mutation directly affects cell wall organization and cell expansion. The shv3-2svl1-1 double mutant has abnormally shaped epidermal cells Besides the phenotypes observed in other cell wall mutants, a number of unique phenotypes were observed in the shv3-2svl1-1 double mutant. Interestingly, the guard cells in shv3-2svl1-1 were larger than those in the wild type (Fig. 6A, B), although the stomatal response to ABA and light was normal (data not shown). When fixed with ethanol and cleared with chloral hydrate, the ventral sides of the guard cells in shv3-2svl1-1 were obviously swollen (Fig. 6C). Another visible phenotype in shv3-2svl1-1 was aberrant swelling of the hypocotyl epidermis (Fig. 6D). Electron microscopic observations revealed that the peak of the swollen site had a rough surface, suggesting 1528 SHV3 and SVLs in cell wall organization B shv3 WT SHV3 SVL1 rRNA rRNA WT B A shv3svl1 shv3svl1 svl1 Area of stomata (µm2) WT A WT shv3svl1 + SVL1 600 500 WT shv3svl1 400 300 200 100 0 shv3svl1 C WT shv3svl1 Phloroglucinol stain D WT shv3svl1 Hypocotyl length (mm) C WT shv3svl1 16 12 8 D 4 0 WT E shv3svl1 VSP1 PDF1.2 5 mm 5 mm rRNA Fig. 5 shv3-2svl1-1 shows features of altered cell wall and cell expansion mutants. (A) RT–PCR analysis of SHV3 and SVL1 expression in each disruptant mutant. Total RNA was extracted from 7-d-old seedlings. Each cDNA fragment was amplified with specific primers. 18S rRNA was used as an internal control. (B) Photographs of the root–shoot junction in wild-type, shv3-2svl11, phloroglucinol-stained shv3-2svl1-1 and phloroglucinol-stained transgenic shv3-2svl1-1 plants with a wild-type SVL1 transgene. Lignified cell walls were stained red by phloroglucinol-HCl in 3-dold shv3-2svl1-1 light-grown seedlings. (C) Dark-grown hypocotyl of 4-d-old wild-type (WT) and shv3-2svl1-1 seedlings. (D) Hypocotyl length of dark-grown 4-d-old WT and shv3-2svl1-1 seedlings. The means SD from 20 samples are shown. (E) Accumulation of transcripts of the jasmonic acid- and ethylene-responsive genes VSP1 and PDF1.2 in shv3-2svl1-1 seedlings. Total RNA was extracted from 7-d-old light-grown seedlings, and each gene was amplified by PCR from an equal quantity of the reverse-transcribed products. 18S rRNA was used as an internal control. an abnormal outer cell surface. These observations suggest that SHV3 and SVL1 play important roles in determining epidermal cell morphology by conferring cell wall stability. Fig. 6 Abnormal epidermal cells in the shv3-2svl1-1 double mutant. (A) Stomata from shv3-2svl1-1 and wild-type plants. Bar ¼ 10 mm. (B) Size of the opened stomata in shv3-2svl1-1, calculated as the product of the long and short axes. Twenty stomata from a single leaf were used in each experiment. The data are the means SD of three independent experiments. The P-value calculated by Student’s t-test was 50.01. (C) Aberrant shape of the shv3-2svl1-1 stomata. An shv3-2svl1-1 leaf fixed with ethanol and cleaned with chloral hydrate showing swelling on the ventral sides of the guard cells. Bar ¼ 20 mm. (D) Aberrant swelling of the shv32svl1-1 hypocotyl epidermis. Electron micrographs of hypocotyls from 7-d-old wild-type (upper left) and shv3-2svl1-1 (upper right and lower) seedlings. Bars ¼ 50 mm (upper left), 100 mm (upper right, bottom left) and 10 mm (bottom right). Characterization of the shv3-2svl1-1 cell wall To characterize the shv3-2svl1-1 cell wall, we isolated and analyzed alcohol-insoluble residues (AIRs) from 4-dold dark-grown seedlings (Table 1). First, we measured the SHV3 and SVLs in cell wall organization Table 1 Composition of the AIR from 4-d-old darkgrown seedlings 0.005 −0.008 −0.004 0 0.004 0.008 PC1 PC1 loading B 0.20 1061 1037 953 1105 0.10 1165 0 −0.10 1661 900 −0.20 1549 1000 crystalline cellulose content. The AIRs of the dark-grown shv3-2svl1-1 seedlings contained about 45% less crystalline cellulose, highlighting the requirement for SHV3 and SVL1 for crystalline cellulose. Secondly, we analyzed the neutral sugar composition of the AIRs. Since excess starch-derived glucose was detected in shv3-2svl1-1, the neutral sugar compositions were compared excluding glucose. Indeed, the AIRs of the dark-grown shv3-2svl1-1 seedlings contained a larger amount of starch. No drastic changes in neutral sugar composition were found, although slightly higher arabinose and slightly lower xylose contents were detected in shv32svl1-1. A shorter dark-grown hypocotyl increased accumulation of ethylene/jasmonic acid-responsive gene transcripts and ectopic lignification were also observed in the cellulosedeficient mutant cev1/eli1. Therefore, it is possible that a reduced cellulose content is the major cause of the phenotypes observed in the shv3-2svl1-1 double mutant. On the other hand, some phenotypes unique to shv3-2svl1-1, such as ruptured root hair cells and swollen guard cells, have not been observed in cellulose-deficient mutants. Hence, the presence of other changes in the cell wall was suspected. To investigate whether shv3-2svl1-1 has an aberrant cell wall composition, we performed Fouriertransform infrared (FTIR) analysis, which provides information on cell wall composition and architecture (McCann et al. 1992). FTIR absorption spectra of the double mutant and wild type were obtained using 4-d-old dark-grown hypocotyls. Principal components analysis using these −0.010 1100 Expressed as mg mg–1 AIR. b Degree of methylesterified uronic acids. c The amount of each neutral sugar is presented as the percentage of the total neutral sugars after deduction of the glucose content. The data are the means SD of three experiments. Indicates a significant difference compared with the wild type (Student’s t-test, P50.05). yIndicates a marginally significant difference from the wild type (Student’s t-test, P50.1). a −0.005 1200 11.5 0.7 4.5 0.2 26.2 2.6 21.5 0.5 4.4 0.4 31.9 1.4 1300 11.3 0.7 4.2 0.2 20.9 1.2 23.8 1.2 4.4 1.3 35.5 1.7 0 1400 84 6 53.6 1.0 85 14y 52 6 1500 154 6 5.8 0.3 63 11 71 6 1600 Cellulose Starcha Uronic acidsa DM%b Neutral sugarsc Rhamnose Fucose Arabinose Xylose Mannose Galactose shv3-2svl1-1 WT shv3svl1 1700 a Wild type A 0.010 PC2 Components 1529 Wave numbers (cm−1) Fig. 7 FTIR analysis of the shv3-2svl1-1 cell wall. (A) FTIR analysis of dark-grown hypocotyl cell walls. Principal components analysis was performed using spectra from 14 shv3-2svl1-1 plants and 12 wild-type plants. The shv3-2svl1-1 and wild-type data were separated using the first principal component (PC1) score, which explains 38.35% of the variance. (B) PC1 loading. Positive peaks characteristic of cellulose (1,037, 1,061, 1,105 and 1,165 cm–1) indicate that the shv3-2svl1-1 cell walls are poorer in cellulose than those of the wild type. The peak at 953 cm–1 corresponds to unesterified pectin. Negative peaks at 1,549 and 1,661 cm–1 indicate that the shv3-2svl1-1 cell walls are enriched in protein relative to the wild type. spectral data revealed a clear separation of shv3-2svl1-1 and wild type in principal component 1 (PC1) (Fig. 7A). The data for PC1 indicated that the cell walls of shv3-2svl1-1 are poorer in cellulose (corresponding to peaks at 1,037, 1,061, 1,105 and 1,165 cm–1) but richer in protein (corresponding to peaks at 1,550 and 1,650 cm–1) than those of the wild type (Wilson et al. 2000) (Fig. 7B), consistent with the results of our AIR analysis. Another remarkable difference was the absorption at 953 cm–1, which reflects the amount of polygalacturonic acid (Synytsya et al. 2003). Because this 1530 SHV3 and SVLs in cell wall organization absorption is diminished by methyl esterification of the carboxyl group (Synytsya et al. 2003), the total uronic acid content in shv3-2svl1-1 may have decreased or the degree of methyl esterification may have increased. To confirm these quantitative changes, the total uronic acid and methyl ester contents were measured. Contrary to our expectations, the cell walls of the dark-grown shv3-2svl1-1 seedlings had an increased amount of uronic acid and a decreased amount of methyl esterification when compared with the wild type (Table 1). The uronic acid content and degree of methyl esterification were not responsible for the change in absorption at 953 cm–1 in our FTIR analysis, suggesting that some other modification or altered interaction may affect the infrared absorption of the pectic polysaccharides in shv3-2svl1-1. These results suggest that the shv3-2svl1-1 mutation affects the properties and amount of the pectins that are present. Increasing the borate concentration partially suppresses the shv3svl1 and shv3 phenotypes We hypothesized that pectin cross-linking is affected by the condition of the pectin. To examine whether promoting pectin cross-linking would suppress the mutant phenotype, shv3-2svl1-1 and shv3 were grown on medium containing an increased concentration of borate, which is supposed to be involved in the cross-linking of pectic domain rhamnogalacturonan-II (RG-II) and other cell wall carbohydrates (Blevins and Lukaszewski 1998). Four-day-old seedlings grown on medium containing 0.1 mM borate were transferred to medium containing a higher concentration of borate and grown for 3 d. A significant increase in the number of unruptured root hairs was observed in both shv3-2svl1-1 and shv3; however, the unruptured root hairs in shv3 were normal (i.e. elongated) in shape (Fig. 8), whereas those in shv3-2svl1-1 were irregular (Supplementary Fig. S5). Under the growth conditions used, 2.5 mM borate was more effective than 0.5 mM borate, indicating dose dependency. A borate concentration of 3.5 mM reduced root hair elongation, presumably due to toxicity. On the other hand, increasing the concentration of calcium, which is involved in the cross-linking of homogalacturonan, did not suppress any of the observed phenotypes (data not shown). These results imply that the stability of RG-II cross-linking is reduced in shv3-2svl1-1 and shv3. Increased borate concentrations did not suppress any of the other phenotypes in the shv3-2svl1-1 double mutant under the same conditions. To confirm whether RG-II cross-linking is altered in the mutant, we assessed the proportion of borate estercross-linked RG-II dimers in shv3-2svl1-1 and the wild type. AIRs purified from seedlings grown on 0.1 mM borate were analyzed. As shown in Fig. 9, the proportion of RG-II dimers was slightly but significantly reduced in shv3-2svl1-1. Borate (mM) 0.1 0.5 2.5 3.5 shv3-2 WT Fig. 8 Suppression of the shv3 phenotype by an increased concentration of borate. Four-day-old seedlings grown on normal medium containing 0.1 mM borate were transferred to medium containing various concentrations (0.1, 0.5, 2.5 or 3.5 mM) of borate and grown for 3 d. Bar ¼ 0.4 mm. Discussion To date, only three mutants with ruptured root hairs have been reported: kjk, lrx1lrx2 and mrh4 (Baumberger et al. 2001, Favery et al. 2001, Jones et al. 2006). KJK encodes a cellulose synthase-like D3 protein predicted to be involved in the synthesis of polysaccharides other than cellulose (Dhugga et al. 2004, Liepman et al. 2005). LRX1 and LRX2, which have leucine-rich repeats and extensinlike domains, regulate cell wall organization in root hairs, although their biochemical functions are unknown (Baumberger et al. 2001, Baumberger et al. 2003, Diet et al. 2006). MRH4 encodes the COBRA-like protein COBL9 (Jones et al. 2006). The ruptured root hair phenotype indicates severe cell wall defects, suggesting that the responsible genes have important roles in cell wall organization. However, to our knowledge, there are no reports of other phenotypes in these mutants, and these genes have not been evaluated for their contributions in other tissues. In this study, we demonstrated that SHV3 has critical roles not only in root hair cells but also in other tissues. Cell wall analysis revealed distinct characteristics of the cell wall components in the shv3-2svl1-1 double mutant, including decreased crystalline cellulose accumulation (Table 1, Fig. 7). This result indicates that SHV3 and SVL1 are novel factors required for the accumulation of crystalline cellulose. A reduced cellulose content has also been reported in mutants of ELI1/CEV1 and PRC1, which are involved in primary cell wall synthesis (Fagard et al. 2000, Cano-Delgado et al. 2003). Although eli1/cev1 and prc1 show more severe growth defects than the shv3-2svl1-1 SHV3 and SVLs in cell wall organization dimer (496s) monomer (536s) W#1 RI (mV) W#2 m#1 m#2 300 500 700 Retention time (s) dimer / (dimer + monomer) wild type 0.908 ± 0.015 shv3svl1 0.859 ± 0.009 Fig. 9 shv3svl1 affects the proportion of cross-linked RG-II dimers. RG-II dimers and monomers solubilized from the AIRs by endopolygalacturonase were separated by size-exclusion HPLC with a refractive index detector. The proportion of RG-II dimers and monomers is expressed as RG-II dimer/(RG-II dimer þ monomer). The data are the means SD of three independent experiments. A representative chromatogram of wild type (W) and shv3svl1 (m) (in duplicate) is shown. The P-value calculated by Student’s t-test was 50.01. double mutant, they do not have ruptured root hairs (Desnos et al. 1996, Cano-Delgado et al. 2000). Therefore, it is likely that abnormalities other than the reduced cellulose content contribute to root hair rupture in the shv3-2svl1-1 mutant. Our methyl esterified pectin assay and FTIR spectroscopic results indicate a reduced rate of methyl esterified pectin formation and other alterations in pectin structure or interactions in the shv3-2svl1-1 double mutant (Fig. 7). We also demonstrated that the mutant had a lower rate of 1531 RG-II dimer formation. Additionally, GFP–SHV3 was localized mainly at the plasma membrane (Fig. 1). These observations suggest that SHV3 and related proteins are involved in pectin network formation at the plasma membrane, rather than biosynthesis. The pectin matrix is essential for mechanical stability of the cell wall during rapid tip growth in pollen tubes (Jiang et al. 2005, Iwai et al. 2006). The many mechanisms common to tip growth in root hairs and pollen tubes suggest that pectins are also essential for tip growth in root hairs (Cole and Fowler 2006). In this study, we demonstrated that the shv3 and shv32svl1-1 root hair phenotype could be suppressed by an increased concentration of borate. Similar characteristics were previously observed in mur1, a fucose-deficient mutant; a high borate concentration (2.6 mM) successfully rescued the reduced tensile strength of the mutant hypocotyls and the reduced formation rate of cross-linked borate dimers (O’Neill et al. 2001, Ryden et al. 2003). In the mur1 mutant, it is reasonable that the altered RG-II is the cause of the mutant phenotype and the effect of borate on RG-II contributes significantly to the rescue of the phenotype, because the effect of mur1 mutation on RG-II is defined and a high concentration of borate is sufficient to rescue the mutant phenotype. In contrast, in the case of the shv3 and shv3svl1 double mutant, the sites of action of borate contributing to the suppression of the root hair phenotype were obscure, because the mutations affect not only RG-II formation, and increasing the borate content was not sufficient to reverse the shv3 and shv3svl1 phenotypes completely. However, there are only a few cell wall-related mutants whose phenotypes are suppressed by an increased concentration of borate, suggesting that this observed borate dependency is an important character of shv3 and shv3svl1. The shv3-2svl1-1 double mutant had abnormal epidermal cells (Fig. 6). Root hair-like cell expansion in the hypocotyl epidermis was previously observed in transgenic plants expressing recombinant GLABRA2 (GL2), which was modified to activate the expression of genes involved in root hair cell differentiation (Ohashi et al. 2003). This implies that Arabidopsis hypocotyl epidermal cells have the ability to form root hair-like structures. SHV3 and SVL1 may negatively regulate such structures by modulating cell wall organization. The shape of the guard cells in the mutants was different from that in the wild type, presumably due to loose ventral walls (Fig. 6C). Adequate guard cell movement is thought to be conferred primarily by guard cell-specific cell wall organization (Majewska-Sawka et al. 2002, Jones et al. 2003). It is likely that SHV3 and SVL1 play some role in the development of the guard cell wall. Consistent with this, considerably stronger SVL1 expression was observed in the guard cells (Fig. 4S). To our knowledge, this guard cell phenotype has not been reported 1532 SHV3 and SVLs in cell wall organization previously. It would be interesting to examine this phenotype in other cell wall-related mutants. Taken together, the defects in SHV3 and SVL1 include abnormal cellulose deposition and pectin network formation, suggesting the function of these proteins in cell wall organization. Given the complex composition of the cell wall and our current lack of understanding of how defects in individual cell wall components affect cell wall organization, it is difficult to determine how SHV3 and SVLs are directly involved in cellulose deposition or pectin network formation without precise demonstrations of their biochemical activities. Several GPI-anchored proteins are essential for cell wall organization in plants (Gillmor et al. 2005). The biochemical and molecular functions of individual GPI-anchored proteins, however, are largely unknown. The GPD-like domain in SHV3 and its related proteins implies their functions in catabolism of glycerophosphodiesters such as glycerophosphocholine. Two types of GPD have been reported in carrot and Arabidopsis. They are vacuolar GPD and cell wall GPD, which are assumed to be involved in membrane degradation and uptake of nutrients, respectively (Van der Rest et al. 2002). If this is the case, the absence of SHV3 and SVLs may initially cause the compositional changes in plasma membrane lipid. Disturbance of the lipid composition of the membrane would affect the membrane transport system and the cellulose biosynthesis, and they in turn would cause abnormal cell wall organization, although there is no report demonstrating the direct effect of the phospholipid composition on the cell wall organization. Alternatively, SHV3 and related proteins may possess completely different enzymatic activity from that of GPD, as suggested by the different domain organization and the distinct amino acid residues at the active center. In addition, if the biochemical activity of SHV3 and its paralogs is the same as that of GPDs, the presence of typical GPDs in the cell wall (Van der Rest et al. 2004) would mask the phenotype of the defect of the SHV3 family. A large number of modifications are required for the arrangement of and interactions between cell wall components. It is possible that SHV3 and its related proteins are involved in their regulation. This idea may be consistent with the notion that homologs of these genes are found only in plants which have developed the unique process of cell wall organization. Determining the exact activity of these proteins will not only allow us to characterize their biochemical properties but will also enhance our understanding of cell wall organization. background) mutant line was obtained from a collection of Ds transposon-tagged lines (Kuromori et al. 2004). The shv3-1 seeds were kindly provided by Dr. Claire Grierson (University of Bristol, UK). The T-DNA insertion lines shv3-2 (SALK_024208), svl1-1 (SALK_064539) and svl2-1 (SALK_057865) (Alonso et al. 2003) were obtained from the Arabidopsis Biological Resource Center. The plants were grown as described previously (Nishimura et al. 2005). The point mutation in shv3-1 was detected by DNA sequencing of the At4g26690 gene in the shv3-1 mutant. To generate the double and triple mutants, single mutants were crossed and the presence of T-DNA insertions in the F2 progeny was confirmed by PCR (Supplementary Table S2). The methods used to isolate genomic DNA and the PCR conditions were described previously (Nishimura et al. 2005). RT–PCR analysis First-strand cDNA was synthesized from 0.8 mg of total RNA pre-treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) using a ReverTra Ace RT–PCR Kit (Toyobo, Tokyo, Japan) with random hexamers according to the manufacturer’s instructions. Semi-quantitative RT–PCR was performed with 1/40 of the first-strand reaction mixture using gene-specific primers (Supplementary Table S2). The PCR conditions were 958C for 90 s followed by 16 or 32 cycles of 958C for 15 s, 558C for 20 s and 728C for 60 s, and a final hold at 728C for 4 min. In total, 32 cycles were used to amplify SHV3, SVL1, SVL2, SVL3, SVL4, SVL5, VSP1 and PDF1.2. 18S rRNA was used as an internal control (16 cycles). Generation and analysis of the transgenic plants To construct GFP::SHV3, sGFP (S65T) was amplified by PCR and inserted in-frame into the NcoI site of SHV3 (RAFL0816-D08, obtained from the RIKEN BioResource Center), and then the fusion gene was inserted downstream of the CaMV 35S promoter in the T-DNA region of the binary vector pMSH1 (Kawasaki et al. 1999). Imaging of the transgenic plants was performed using an LSM510 confocal microscope (Carl Zeiss, Jena, Germany). For the promoter::GUS constructs, 1.9, 1.5 and 1.5 kb of the putative promoter regions of SHV3, SVL1 and SVL2, respectively, were amplified by PCR using the primers listed in Supplementary Table S2, and inserted upstream of the GUS gene in the T-DNA region of the binary vector pBI101. Thin sections of stained tissues were prepared using a Technovit 7100 Plastic Embedding Kit (Kulzer, Wehrheim, Germany). For complementation of the shv3-2svl1-1 mutation, a genomic DNA fragment containing the SVL1 coding region (1.5 kb upstream and 0.5 kb downstream) was amplified by PCR and cloned into the T-DNA region of the binary vector pGreenII, and then introduced into the shv3-2svl1-1 double mutant. The M2 and M3 progeny were used for characterization. Materials and Methods Phloroglucinol staining Phloroglucinol staining was performed as described by CanoDelgado et al. (2000). The plants were cleaned with ethanol then mounted in a 2% phloroglucinol-HCl solution. Lignin staining was observed in seedlings embedded in chloral hydrate : glycerol : water (8 : 1 : 2). Plant materials and growth conditions Arabidopsis thaliana (L) Heynh. ecotypes Columbia (Col) and Nossen (Nos) were used in this study. The shv3-3 (15-1096-1, Nos Cell wall characterization FTIR analysis of dark-grown hypocotyls was performed as described by Fagard et al. (2000). Four-day-old dark-grown SHV3 and SVLs in cell wall organization seedlings were pressed onto a barium fluoride window then rinsed with water. The samples were subsequently dried at room temperature for 3 h. Infrared spectra were collected using a PerkinElmer AutoIMAGE FT-IR Microscope System attached to a Spectrum One FT-IR Spectrometer (PerkinElmer, Shelton, CT, USA) from the middle region of the hypocotyl, avoiding the central cylinder, with a 20 mm 40 mm aperture. All data sets were corrected for the baseline and normalized. Principal components analysis was performed using Win-Discrim software (E. K. Kemsley, Institute of Food Research, Norwich, UK). To prepare the AIRs, plant tissues were collected and ground in liquid nitrogen then washed with 80% ethanol, 95% ethanol, 99.5% ethanol, chloroform : methanol (1 : 1) and acetone, and airdried. To measure the crystalline cellulose content, the AIRs were treated with acetic acid : nitric acid : water (8 : 1 : 2) for 1 h at 1008C, then the sugar content of the insoluble materials was measured with anthrone reagent as described by Updegraff (1969). Avicel PH-101 (Fluka, Bruchs, Switzerland) was used to generate a standard curve. The neutral sugar composition of the AIRs was determined by gas chromatography–mass spectrometry (GC-MS) of the alditol acetate derivatives (York et al. 1985). To measure the total uronic acid content, the AIRs were treated with trifluoroacetic acid and the soluble fraction was used. Determination of the uronic acid content was performed using sulfamic acid and m-hydroxybiphenyl reagent as described by Filisetti-Cozzi and Carpita (1991). D-Glucuronic acid was used to generate a standard curve. The presence of uronosyl methyl esters was determined by saponification according to the method of Wood and Siddiqui (1971). The degree of methyl esterification was calculated from paired assays of the total uronic acids. Starch in the AIRs was detected enzymatically using an F-Kit (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions. Determination of the ratio of borate ester cross-linked RG-II dimers to RG-II monomers was performed as described by Matsunaga and Ishii (2006). Scanning electron microscopy Seedlings (7-d-old), frozen in liquid nitrogen, were attached to the stage of a JSM5610-LV electron microscope (JEOL, Tokyo, Japan) and observed under high-vacuum conditions according to the manufacturer’s instructions. Supplementary material Supplementary material are available at PCP Online. Funding Grant-in-Aid from the Ministry of Education, Sports, Culture, Science and Technology (15570045, 20570050 to T.H.), Japan; RIKEN President’s Special Research Grant (T.H.). Acknowledgments We thank Drs. Claire Grierson and Miki Fujita for providing the shv3-1 seeds and plasmids, respectively, and Dr. Chieko Saito for helping in microscopic analysis using the confocal microscope. We also thank the Arabidopsis Biological Resource Center and 1533 RIKEN BioResource Center for the T-DNA insertion lines and a cDNA clone. References Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657. Baumberger, N., Ringli, C. and Keller, B. (2001) The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana. Genes Dev. 15: 1128–1139. Baumberger, N., Steiner, M., Ryser, U., Keller, B. and Ringli, C. (2003) Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development. Plant J. 35: 71–81. Blevins, D.G. and Lukaszewski, K.M. (1998) Boron in plant structure and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 481–500. Borner, G.H., Lilley, K.S., Stevens, T.J. and Dupree, P. (2003) Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132: 568–577. Borner, G.H., Sherrier, D.J., Stevens, T.J., Arkin, I.T. and Dupree, P. (2002) Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis. Plant Physiol. 129: 486–499. Brocard-Gifford, I., Lynch, T.J., Garcia, M.E., Malhotra, B. and Finkelstein, R.R. (2004) The Arabidopsis thaliana ABSCISIC ACIDINSENSITIVE8 encodes a novel protein mediating abscisic acid and sugar responses essential for growth. Plant Cell 16: 406–421. Cano-Delgado, A.I., Metzlaff, K. and Bevan, M.W. (2000) The eli1 mutation reveals a link between cell expansion and secondary cell wall formation in Arabidopsis thaliana. Development 127: 3395–3405. Cano-Delgado, A., Penfield, S., Smith, C., Catley, M. and Bevan, M. (2003) Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. Plant J. 34: 351–362. Cole, R.A. and Fowler, J.E. (2006) Polarized growth: maintaining focus on the tip. Curr. Opin. Plant Biol. 9: 579–588. Cosgrove, D.J. (2005) Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 6: 850–861. Desnos, T., Orbovic, V., Bellini, C., Kronenberger, J., Caboche, M., Traas, J. and Hofte, H. (1996) Procuste1 mutants identify two distinct genetic pathways controlling hypocotyl cell elongation, respectively in dark- and light-grown Arabidopsis seedlings. Development 122: 683–693. Dhugga, K.S., Barreiro, R., Whitten, B., Stecca, K., Hazebroek, J., Randhawa, G.S., Dolan, M., Kinney, A.J., Tomes, D., Nichols, S. and Anderson, P. (2004) Guar seed b-mannan synthase is a member of the cellulose synthase super gene family. Science 303: 363–366. Diet, A., Link, B., Seifert, G.J., Schellenberg, B., Wagner, U., Pauly, M., Reiter, W.D. and Ringli, C. (2006) The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase. Plant Cell 18: 1630–1641. Ellis, C., Karafyllidis, I., Wasternack, C. and Turner, J.G. (2002) The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell 14: 1557–1566. Fagard, M., Desnos, T., Desprez, T., Goubet, F., Refregier, G., Mouille, G., McCann, M., Rayon, C., Vernhettes, S. and Höfte, H. (2000) PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis. Plant Cell 12: 2409–2424. Favery, B., Ryan, E., Foreman, J., Linstead, P., Boudonck, K., Steer, M., Shaw, P. and Dolan, L. (2001) KOJAK encodes a cellulose synthase-like protein required for root hair cell morphogenesis in Arabidopsis. Genes Dev. 15: 79–89. Filisetti-Cozzi, T.M. and Carpita, N.C. (1991) Measurement of uronic acids without interference from neutral sugars. Anal. Biochem. 197: 157–162. Gillmor, C.S., Lukowitz, W., Brininstool, G., Sedbrook, J.C., Hamann, T., Poindexter, P. and Somerville, C. (2005) Glycosylphosphatidylinositolanchored proteins are required for cell wall synthesis and morphogenesis in Arabidopsis. Plant Cell 17: 1128–1140. 1534 SHV3 and SVLs in cell wall organization Iwai, H., Hokura, A., Oishi, M., Chida, H., Ishii, T., Sakai, S. and Satoh, S. (2006) The gene responsible for borate cross-linking of pectin rhamnogalacturonan-II is required for plant reproductive tissue development and fertilization. Proc. Natl Acad. Sci. USA 103: 16592–16597. Jiang, L., Yang, S.L., Xie, L.F., Puah, C.S., Zhang, X.Q., Yang, W.C., Sundaresan, V. and Ye, D. (2005) VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. Plant Cell 17: 584–596. Jones, L., Milne, J.L., Ashford, D. and McQueen-Mason, S.J. (2003) Cell wall arabinan is essential for guard cell function. Proc. Natl Acad. Sci. USA 100: 11783–11788. Jones, M.A., Raymond, M.J. and Smirnoff, N. (2006) Analysis of the root hair morphogenesis transcriptome reveals the molecular identity of six genes with roles in root hair development in Arabidopsis. Plant J. 45: 83–100. Kawasaki, T., Henmi, K., Ono, E., Hatakeyama, S., Iwano, M., Satoh, H. and Shimamoto, K. (1999) The small GTP-binding protein rac is a regulator of cell death in plants. Proc. Natl Acad. Sci. USA 96: 10922–10926. Kuromori, T., Hirayama, T., Kiyosue, Y., Takabe, H., Mizukado, S., Sakurai, T., Akiyama, K., Kamiya, A., Ito, T. and Shinozaki, K. (2004) A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. Plant J. 37: 897–905. Kuromori, T., Wada, T., Kamiya, A., Yuguchi, M., Yokouchi, T., et al. (2006) A trial of phenome analysis using 4000 Ds-insertional mutants in gene-coding regions of Arabidopsis. Plant J. 47: 640–651. Lalanne, E., Honys, D., Johnson, A., Borner, G.H., Lilley, K.S., Dupree, P., Grossnicklaus, U. and Twell, D. (2004) SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis. Plant Cell 16: 229–240. Larson, T.J., Ehrmann, M. and Boos, W. (1983) Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon. J. Biol. Chem. 258: 5428–5432. Li, Y., Jones, L. and McQueen-Mason, S. (2003) Expansins and cell growth. Curr. Opin. Plant Biol. 6: 603–610. Liepman, A.H., Wilkerson, C.G. and Keegstra, K. (2005) Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases. Proc. Natl Acad. Sci. USA 102: 2221–2226. Majewska-Sawka, A., Munster, A. and Rodriguez-Garcia, M.I. (2002) Guard cell wall: immunocytochemical detection of polysaccharide components. J. Exp. Bot. 53: 1067–1079. Matsunaga, T. and Ishii, T. (2006) Borate cross-linked/total rhamnogalacturonan II ratio in cell walls for the biochemical diagnosis of boron deficiency in hydroponically grown pumpkin. Anal. Sci. 22: 1125–1127. McCann, M.C., Hammouri, M., Wilson, R., Belton, P. and Roberts, K. (1992) Fourier transform infrared microspectroscopy is a new way to look at plant cell walls. Plant Physiol. 100: 1940–1947. Nicol, F., His, I., Jauneau, A., Vernhettes, S., Canut, H. and Hofte, H. (1998) A plasma membrane-bound putative endo-1,4-b-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis. EMBO J. 17: 5563–5576. Nishimura, N., Kitahata, N., Seki, M., Narusaka, Y., Narusaka, M., Kuromori, T., Asami, T., Shinozaki, K. and Hirayama, T. (2005) Analysis of ABA hypersensitive germination2 revealed the pivotal functions of PARN in stress response in Arabidopsis. Plant J. 44: 972–984. O’Neill, M.A., Eberhard, S., Albersheim, P. and Darvill, A.G. (2001) Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294: 846–849. Ohashi, Y., Oka, A., Rodrigues-Pousada, R., Possenti, M., Ruberti, I., Morelli, G. and Aoyama, T. (2003) Modulation of phospholipid signaling by GLABRA2 in root-hair pattern formation. Science 300: 1427–1430. O’Neill, M.A., Ishii, T., Albersheim, P. and Darvill, A.G. (2004) Rhamnogalacturonan II: structure and function of a borate cross-linked cell wall pectic polysaccharide. Annu. Rev. Plant Biol. 55: 109–139. Pagant, S., Bichet, A., Sugimoto, K., Lerouxel, O., Desprez, T., McCann, M., Lerouge, P., Vernhettes, S. and Höfte, H. (2002) KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis. Plant Cell 14: 2001–2013. Page, R.D. (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: 357–358. Parker, J.S., Cavell, A.C., Dolan, L., Roberts, K. and Grierson, C.S. (2000) Genetic interactions during root hair morphogenesis in Arabidopsis. Plant Cell 12: 1961–1974. Penninckx, I.A., Thomma, B.P., Buchala, A., Metraux, J.P. and Broekaert, W.F. (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103–2113. Ridley, B.L., O’Neill, M.A. and Mohnen, D. (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57: 929–967. Rojo, E., Leon, J. and Sanchez-Serrano, J.J. (1999) Cross-talk between wound signaling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J. 20: 135–142. Roudier, F., Fernandez, A.G., Fujita, M., Himmelspach, R., Borner, G.H., Schindelman, G., Song, S., Baskin, T.I., Dupree, P., Wasteneys, G.O. and Benfey, P.N. (2005) COBRA, an Arabidopsis extracellular glycosylphosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation. Plant Cell 17: 1749–1763. Ryden, P., Sugimoto-Shirasu, K., Smith, A.C., Findlay, K., Reiter, W.D. and McCann, M.C. (2003) Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II–borate complexes. Plant Physiol. 132: 1033–1040. Scheible, W.R. and Pauly, M. (2004) Glycosyltransferases and cell wall biosynthesis: novel players and insights. Curr. Opin. Plant Biol. 7: 285–295. Schindelman, G., Morikami, A., Jung, J., Baskin, T.I., Carpita, N.C., Derbyshire, P., McCann, M.C. and Benfey, P.N. (2001) COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis. Genes Dev. 15: 1115–1127. Sedbrook, J.C., Carroll, K.L., Hung, K.F., Masson, P.H. and Somerville, C.R. (2002) The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth. Plant Cell 14: 1635–1648. Shi, H., Kim, Y., Guo, Y., Stevenson, B. and Zhu, J.K. (2003) The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion. Plant Cell 15: 19–32. Synytsya, A., Copikova, J., Matejka, P. and Machovic, V. (2003) Fourier transform Raman and infrared spectroscopy of pectins. Carbohyd. Polym. 54: 97–106. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882. Tommassen, J., Eiglmeier, K., Cole, S.T., Overduin, P., Larson, T.J. and Boos, W. (1991) Characterization of two genes, glpQ and ugpQ, encoding glycerophosphoryl diester phosphodiesterases of Escherichia coli. Mol. Gen. Genet. 226: 321–327. Updegraff, D.M. (1969) Semi-micro determination of cellulose in biological materials. Anal. Biochem. 32: 420–424. Van Der Rest, B., Boisson, A.M., Gout, E., Bligny, R. and Douce, R. (2002) Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase. Plant Physiol. 130: 244–255. Van Der Rest, B., Rolland, N., Boisson, A.M., Ferro, M., Bligny, R. and Douce, R. (2004) Identification and characterization of plant glycerophosphodiester phosphodiesterase. Biochem. J. 379: 601–607. Vissenberg, K., Van Sandt, V., Fry, S.C. and Verbelen, J.P. (2003) Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays. J. Exp. Bot. 54: 335–344. SHV3 and SVLs in cell wall organization Wilson, R.H., Smith, A.C., Kacurakova, M., Saunders, P.K., Wellner, N. and Waldron, K.W. (2000) The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy. Plant Physiol. 124: 397–405. Wood, P.J. and Siddiqui, I.R. (1971) Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity. Anal. Biochem. 39: 418–428. 1535 York, W.S., Darvill, A.G., McNeil, M., Stevenson, T.T. and Albersheim, P. (1985) Isolation and characterization of plant cell walls and cell wall constituents. Methods Enzymol. 118: 3–40. Zhong, R., Kays, S.J., Schroeder, B.P. and Ye, Z.H. (2002) Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene. Plant Cell 14: 165–179. (Received June 6, 2008; Accepted August 15, 2008)