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First published online April 22, 2005; 10.1104/pp.104.058248 Plant Physiology 138:232-242 (2005) © 2005 American Society of Plant Biologists
A Novel Short-Root Gene Encodes a Glucosamine-6-Phosphate Acetyltransferase Required for Maintaining Normal Root Cell Shape in Rice1State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310029, People's Republic of China
Glycosylation is a posttranslational modification occurring in many secreted and membrane-associated proteins in eukaryotes. It plays important roles in both physiological and pathological processes. Most of these protein modifications depend on UDP-N-acetylglucosamine. In this study, a T-DNA insertional rice (Oryza sativa) mutant exhibiting a temperature-sensitive defect in root elongation was isolated. Genetic and molecular analysis indicated that the mutated phenotype was caused by loss of function of a gene encoding a glucosamine-6-P acetyltransferase (designated OsGNA1), which is involved in de novo UDP-N-acetylglucosamine biosynthesis. The aberrant root morphology of the gna1 mutant includes shortening of roots, disruption of microtubules, and shrinkage of cells in the root elongation zone. Our observations support the idea that protein glycosylation plays a key role in cell metabolism, microtubule stabilization, and cell shape in rice roots.
UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite that plays an important role in protein and lipid glycosylation in eukaryotes. The glycans on cell surface glycoproteins are instrumental for cell-cell or cell-matrix interactions, immune reactions, and tumor development, while sugar modifications on secreted proteins are important for their transport, biological activity, and clearance from circulation (Varki et al., 1999  ). Asn (N)-linked glycans (N-glycans) are components of most membrane-associated and secreted proteins in eukaryotic cells. N-glycans are assembled by the initial step of the transfer of a synthesized dolichol-linked oligosaccharide precursor en bloc to a nascent polypeptide chain in the endoplasmic reticulum. Maturation of the oligomannose precursor requires the action of glucosidases, mannosidases, and Golgi-resident glycosyltransferases. Synthesis of this oligosaccharide precursor initiates with the donor of UDP-GlcNAc and the enzyme of UDP-GlcNAc: dolichol phosphate GlcNAc-1-P transferase (Lowe and Marth, 2003; Haltiwanger and Lowe, 2004). The other proteins that pass through the secretory apparatus are O-glycan modified by the addition of carbohydrates to the OH group of Ser or Thr. The synthesis of O-glycans is initiated with N-acetylgalactosamine (GalNAc) modification of Ser or Thr and employs sequential addition of monosaccharides. The branch synthesis of many O-glycans requires Golgi-routed UDP-GlcNAc (for review, see Lowe and Marth, 2003).
UDP-GlcNAc also involves the generation of glycosylphospatidylinositol linkers for anchoring a variety of cell surface molecules to the plasma membrane. The transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol is the initial reaction of the glycosylphospatidylinositol assembly (Lowe and Marth, 2003
In plants, glycans modulate many biological processes, such as somatic embryogenesis (van Hengel et al., 2001
Generation of UDP-GlcNAc is possible by both de novo synthesis and salvage pathways, and the respective enzymes have been identified (for review, see Schachter, 1978 In this study, we identified and characterized a temperature-sensitive short-root mutant in a T-DNA insertional rice (Oryza sativa) library. The genetic and molecular analyses indicated that a yeast (Saccharomyces cerevisiae) GNA1 homology gene was disrupted by the insertion of T-DNA in this mutant. Downstream routes emanating from UDP-GlcNAc in the gna1 mutant were affected differentially with predominant aberrations in O-GlcNAc modification and partly in N-glycan modification of cellular proteins. Our results support the idea that protein glycosylation plays an important role in root cell metabolism and microtubule (MT) stabilization in rice.
Isolation of the Short-Root Rice Mutant in Rice
To study the genetic mechanism controlling root growth in plants, we screened a rice T-DNA insertional library (Chen et al., 2003
The defect in root elongation of the NB208 mutant proved to be temperature sensitive. The root lengths of 1-week-old mutant seedlings were about 70% of those of wild-type seedlings at 32°C (Fig. 1, C and D), while showing very short roots at 25°C (Fig. 1E). The defect in root elongation at 25°C could be restored when seedlings were moved to 32°C (Fig. 1F).
Two flanking sequences of the left border and right border of the T-DNA tag were obtained using the plasmid rescue strategy and the thermal asymmetric interlaced (TAIL)-PCR method (Manuel et al., 1980
Northern-blot analysis and RNA in situ hybridization indicate that the OsGNA1 gene was mainly expressed in roots, particularly in the elongation zone of roots in wild-type plants, whereas OsGNA1 transcripts were scarcely detected in the NB208 mutant roots (Fig. 3, A and B). This result indicates that NB208 is a GNA1 null mutant. OsGNA1 cDNA was cloned into the binary vector pCAMBIA2301. The cauliflower mosaic virus 35S promoter-driven OsGNA1 was then introduced into gna1 mutant plants via Agrobacterium tumefaciens-mediated transformation. The root length of nine independent T2 families (20–50 seedlings each) was evaluated. A 3:1 segregation ratio of wild-type to short-root mutants was observed among two independent T2 families. OsGNA1 overexpression in transgenic plants was determined by northern-blot analysis (Fig. 3C). Southern-blot analysis showed that these two transgenic events contain a single copy of the insertion (Fig. 3D). These results confirmed that overexpression of OsGNA1 complemented the short-root phenotype. Thus, the mutant is designated gna1.
Structural and Functional Characterization of OsGNA1
The OsGNA1 gene contains an open reading frame of 165 amino acids. The C terminus of the OsGNA1 polypeptide matches the AT motifs (Mio et al., 1999
To verify whether OsGNA1 can acetylate GlcN-6-P, bacterium-expressed His-OsGNA1 (Fig. 5A) and a control protein, His-S-tag/thrombin, were purified under identical conditions. With 0.1 µg of purified protein, 200 µM of acetyl-CoA, and different amounts of GlcN-6-P per reaction, His-OsGNA1 exerted GlcN-6-P AT activity, while the control proteins did not (Fig. 5B). Since GlcN-1-P did not lead to the release of CoA from acetyl-CoA in the assay, OsGNA1 prefers to use GlcN-6-P as a substrate (Fig. 5C). The average Michaelis-Menten Km value for GlcN-6-P and acetyl-CoA are 145 and 180 µM, respectively. Therefore, it is likely that OsGNA1 exerts GlcN-6-P AT activity but not GlcN-1-P AT activity.
Analysis of UDP-GlcNAc steady-state levels in roots showed that the level of UDP-GlcNAc in gna1 mutants was less than 10% of that in wild-type plants (Table I; Fig. 6, A and B), suggesting that the GNA1-dependent pathway is the major source of UDP-GlcNAc in rice roots. After incubation for 6 h with GlcN, the UDP-GlcNAc level increased in the roots of wild-type plants, but not in the roots of the gna1 mutant (Table I; Fig. 6C). The results indicate that GlcN-6-P AT activity is impaired in gna1 root cells.
Glycosylation in gna1 Mutant Roots To determine whether endoplasmic reticulum/Golgi-mediated glycosylation is impaired in root cells of the gna1 mutant, extracts of root cells were subjected to immunoblot analysis using the peroxidase-conjugated lectins, concanavalin A (ConA) and wheat germ agglutinin (WGA). ConA is used to bind to branches of oligomannose chains on N-glycoproteins, while WGA recognizes salic acid and GlcNAc on N- and O-linked oligosaccharides. The results showed that the amount of higher molecular weight glycoproteins was decreased dramatically in mutant roots compared to wild-type roots for lectin ConA. No significant difference in the overall amount of glycoproteins between the gna1 mutant and the wild-type root cells was detected for WGA used (Fig. 6D).
To assess the extent of cytosolic O-linked GlcNAc modifications of proteins in the gna1 mutant, cytosolic extracts were labeled with UDP-[3H]Gal using
To investigate the relationship between glycoproteins and root elongation in rice, physiological and anatomical analyses were performed. Triphenyltetrazolium chloride (TTC) reduction has been widely used as a general measurement of vitality in plant tissues. In plant tissues, TTC is mainly reduced by dehydrogenase enzymes, most of which are associated with mitochondrial function (Comas et al., 2000
Feulgen staining, which selectively stains DNA, has been widely used as a measure of DNA content and reveals distinctive tissue with high DNA content (Demchenko et al., 2004 ). A red region in the meristematic zone was observed in wild-type root tips after Feulgen stain (Fig. 7C) but not in mutant root tips (Fig. 7, D and E). Compared with wild-type roots, the cell division region of the mutant roots is smaller when grown at room temperature for 2 and 5 d (Fig. 7E), indicating that cell division in the mutant roots was inhibited.
Evans blue dye staining of cells with a completely permeable plasma membrane is used to identify the dead cells (Gaff and Okong'O-gola, 1971
Immunolocalization of cortical MTs was performed to examine their status in the mutant plants. In wild-type root tips, cortical MTs at the elongation zone were oriented transversely along the elongation axis (Fig. 7H), whereas no cortical MT bundles were detected in gna1 roots grown at room temperature. The disrupted MTs associated with the plasma membrane exhibited condensed spots in the cortex cells spreading into the endodermal cells of gna1 roots (Fig. 7I). The gna1 root cells grown at high temperature (32°C), however, showed normal cortical MTs (Fig. 7J).
To observe cell size and shape, longitudinal and transverse sections were prepared from root maturation, elongation, and meristem zones. Roots of wild-type seedlings exhibited highly organized cell files and defined cell shapes after 2 d of growth at room temperature (Fig. 8A). The root tips of gna1 seedlings at the same temperatures displayed abnormal cell shape and length under the same growth conditions (Fig. 8, B and C). From the epidermis to the cortical layer, the mutant root cells were shrunken and the adjacent cell walls were inlaid on each other in maturation, elongation, and meristem zones (Fig. 8, E, G, and I), compared with wild-type roots (Fig. 8, D, F, and H). The stele cells also became distorted after 5 d of growth at room temperature (Fig. 8F).
Using a transmission electron microscope (H-600; Hitachi, Tokyo), it was observed that the endodermis, cortex, and stele cells of the gna1 root were characterized by cytoplasmic shrinkage and distorted cell shape after being grown at room temperature (Fig. 9, A, C, E, and F). Further examination of the shrunken cells revealed that organelles in the cells were disaggregated and empty. The nucleolus and nuclear membrane were intact before the cell became empty, although the chromatin in the nuclei had been disaggregated (Fig. 9, C and D).
A novel short-root gene for a GlcN-6-P AT in rice (OsGNA1) was identified from a T-DNA insertional rice mutant library. Our results indicate that OsGNA1 is required for maintaining normal root cell metabolism and cell shape. Northern-blot analysis and RNA in situ hybridization revealed that the OsGNA1 is expressed preferentially in the root elongation zone, suggesting its participation in UDP-GlcNAc synthesis in roots.
Using purified recombinant His-tagged OsGNA1 expressed in Escherichia coli (Fig. 5A), we determined that the Michaelis-Menten Km value for GlcN-6-P and acetyl-CoA was 145 and 180 µM, respectively, which is comparable to the Km value of ScGNA1 for GlcN-6-P and acetyl-CoA (124.13 and 228.78 µM). These results support the idea that the AT motif and the enzymatic character of the GNA family were conserved in yeast, animals, and plants (Mio et al., 1999
The limited UDP-GlcNAc level in root cells of the gna1 mutant was insufficient to provide UDP-GlcNAc for protein N-glycosylation (Fig. 6). N-Glycosylation is essential for the proper folding, targeting, and functioning of secreted proteins. N-Glycoproteins play important roles in plant developmental processes, including cell wall synthesis (Lerouge et al., 1998
MTs take part in governing the formation of cell walls and, consequently, cell shape in plants. Many MT-associated proteins regulate the organization and dynamics of MTs in cells. In animals, MT-binding proteins, such as Tau, MAP2, and MAP4, are modified by O-GlcNAc. It has been suggested that acetylation increases the stability of MTs. The acetylation of rabbit skeletal troponin C increases its ability to interact with other troponin components (Maruta et al., 1986 Root length is determined by cell division in the root meristem and cell elongation in the root elongation zone. Our observations suggest that the gna1 mutant primarily reduced cell elongation in the elongation zone and above by causing cell shrinkage (Fig. 8). The effects on cell division in the mutant may be secondary to the effects on elongating cells. Perhaps cell-cell signaling between the cells in the elongation zone and the meristem are required for meristem maintenance, which is disrupted in the mutant. The reduction of respiration in the mutant may result from the disaggregation of the organelle. Cell shrinkage in root cells of the gna1 mutant may be attributed to the reduction of respiration metabolism and the disruption of MTs. The reduced respiration results in the lack of energy to support ion absorption to maintain cell osmotic potential and prevent loss of cellular water. The disrupted MT could not support the cell shape and intracellular transportation. It is possible that the loss of function of many unknown essential proteins, such as ion transporters and cellulose synthase, due to the lack of glycosylation, could also be important in determining cell shrinkage. Proteomic analysis is required to identify the glycosylation-modified proteins, especially the O-GlcNAc-modified proteins related to the defect in the gna1 mutant at room temperature. The collection of O-GlcNAc-modified proteins would greatly facilitate the elucidation of the regulatory mechanism of responding proteins and the specific consensus sequence directing the O-GlcNAc modification.
Mutant Screening
One rice (Oryza sativa) short-root mutant, designated NB208 (Nipponbare containing a bar gene marker), was isolated from about 1,000 T-DNA insertion-mutagenized lines (Chen et al., 2003
DNA fragments flanking the T-DNA insertions were isolated using plasmid rescue strategy (Manuel et al., 1980
BLAST analysis indicates that the gene obtained from the NB208 mutant encodes a putative GlcN-6-P AT (OsGNA1) on chromosome 9. The OsGNA1 cDNA was cloned into a pUC-T vector by reverse transcription-PCR using the primers of 5'-AACCGAAAACCCTAACTGAGTACG-3' and 5'-TCCATCCATACAAGGAGCAATAAC-3'. For the complementation test, the OsGNA1 cDNA clone with the complete CDS was digested with KpnI and XbaI and subcloned into the corresponding site of pCAMBIA2301. The cauliflower mosaic virus 35S promoter was then inserted by EcoRI/SacI sites. Agrobacterium strain EHA105 harboring the above vector was used to transfer the mutant calli via Agrobacterium tumefaciens-mediated transformation (Chen et al., 2003
Five micrograms of genomic DNA was digested with appropriate restriction enzymes, separated on a 0.8% agarose gel, and blotted onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The probe was prepared using a random priming method. Hybridization and washing were conducted according to the manufacturer's instructions. After washing, the blots were analyzed using a Typhoon-8600 phosphorimager system. Twenty micrograms of total RNA was isolated using Trizol (Gibco-BRL, Cleveland) following the manufacturer's protocol, separated on 1% agarose gels containing 1x MOPS and 0.66 M formaldehyde, and transferred onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The probe was prepared using a random priming method and was hybridized following the manufacturer's instructions.
RNA in situ hybridization was performed as described by Harding et al. (2002)
To make the OsGNA1::6-His fusion, the OsGNA1 gene was amplified by PCR using the primers of 5'-AAAAAACATATGGAACAGCCGCTCCCCAC-3' and 5'-AAAAAACTCGAGGAAGTAGAGCCCCATCTGGACG-3'. The reconstructed sequences of OsGNA1 were subcloned into the NdeI and XhoI sites of pET-29b (Novagen, Madison, WI) and defined as pOsGNA1. pOsGNA1 and pET-29b (negative control) were transformed into Escherichia coli BL21 (DE3; Stratagene, La Jolla, CA), and grown on Luria-Bertani medium. Overnight cultures were inoculated into fresh medium at a 1:100 dilution and grown at 37°C until the density was 0.6 A600/mL, and 0.5 mM IPTG was added to the cultures, followed by another 5 h of incubation at 28°C. The cells were collected by centrifugation, suspended in one-twentieth culture volume of sonication buffer (50 mM Tris-acetate, 10 mM EDTA, and 5 mM DTT, pH 7.5), and sonicated on ice for 4 min (1 s on, 2 s off). Cell lysates were centrifuged at 15,000g for 30 min and analyzed using SDS-PAGE. The OsGNA1-6-His fusion proteins were purified under natural conditions using a Ni-NTA column according to the manufacturer's instructions (Qiagen, Hilden, Germany).
The enzyme assay was performed according to the description by Mio et al. (1999)
Proteins were separated by SDS-PAGE using a 7.5% acrylamide gel and electroblotted onto a polyvinylidene difluoride membrane (Amresco, Solon, OH). Blots were incubated in Tris-buffered saline (TBS) plus Tween (TBST; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% [v/v] Tween 20) solution containing 3% (w/v) bovine serum albumin for 60 min at 30°C, and further incubated for 6 h at 4°C with peroxidase-conjugated ConA and WGA diluted with TBST solution containing 1% (w/v) bovine serum albumin. The membranes were washed three times in TBST for 10 min each, and rinsed with TBS for 5 min. The reaction was performed in the reaction buffer with 10 mL 100 mM Tris-HCl, pH 7.5, 100 µL 40 mg/mL diaminobenzidine, 25 µL 80 mg/mL NiCl2, and 15 µL 3% H2O2.
UDP-GlcNAc in distilled water (U4375; Sigma) has a high absorption at 250 to 260 nm and peaks at 254 nm, while GlcN-6-P (G5509; Sigma) also has a high absorption at 250 to 280 nm according to wavelength-scanning analysis. The samples were quantified using a UV spectrophotometer at 254 nm. The retention time and the peaks of UDP-GlcNAc and GlcN-6-P were determined with 1 µg mL–1 UDP-GlcNAc and 10 µg mL–1 GlcN-6-P in distilled water and the root extract, respectively. For rice samples, 5 g of fresh roots were homogenized with a mortar and pestle in 10 mL of distilled water. The homogenate was centrifuged at 12,000g for 10 min at 4°C. The pellet was washed twice with 10 mL of distilled water. The supernatant was filtered with an 0.2-µm membrane and vacuum dried. The dried filtrate was dissolved in 0.2 mL of distilled water and referred to as the nucleotide-sugars extract used for HPLC analysis (Agilent 1100; Agilent Technologies, Palo Alto, CA). Reverse-phase HPLC was performed on a Synchropak WAX anion-exchange column (4.6 x 250 mm; Agilent Technologies), eluted with a concave gradient of ammonium phosphate from 15 mM, pH 3.8, to 1 mM, pH 4.5, over 45 min at a flow rate of 0.8 mL min–1. The operations were repeated three times. The detector signal was recorded and integrated by a personal computer and a chromatography software program (Chemistation 2170AA; Agilent).
The root tips, hairs, and surface were examined by a Leica MZ95 stereomicroscope with a color CCD camera. To examine the DNA content in the root meristem, Feulgen staining was used according to Demchenko et al. (2004) For light and electromicroscopic analysis, root tips were fixed overnight at 4°C in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, and washed three times for 30 min in the same buffer. Root samples were then refixed for 4 h in 1% OsO4 in 0.1 M sodium phosphate buffer, pH 7.2, and washed for 30 min in the same buffer. The samples were dehydrated in a gradient ethanol and embedded in Spurr resin. Semithin sections (3 µm thick) were made using glass knives on a Power Tome XL (RMC-Boeckeler Instruments, Tucson, AZ) microtome and stained in 0.1% methylene blue for 3 to 5 min at 70°C. The samples were rinsed with distilled water and visualized with a Zeiss Axiovert 200 microscope with a color CCD camera (Zeiss). For ultrathin sections, root tips were fixed in 2.5% glutaraldehyde and 1% OsO4, dehydrated in an acetone series, and embedded in Spurr resin. Ultrathin sections (0.1 µm thick) were stained with uranyl acetate for 45 min at room temperature and observed with an electron microscope.
The protocol used for fixing and immunostaining seedling roots has been described (Vitha et al., 1997 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY722189. Received December 14, 2004; returned for revision February 25, 2005; accepted March 15, 2005.
1 This work was supported by the Natural Science Foundation of China (project no. 30230220) and the Bureau of Science and Technology of Zhejiang Province, China.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058248. * Corresponding author; e-mail clspwu{at}zju.edu.cn; fax 86–571–86971323.
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TOP
ABSTRACT
RESULTS
glucosamine-6-P (GlcN-6-P) 
-DNA size markers are indicated on the left. C, Southern blot of GNA1/GNA1 (+/+), GNA1/gna1 (+/–), and gna1/gna1 (–/–) using the OsGNA1 probe. The DNA was digested with EcoRI.
-1,4-galactosyltransferase-mediated [3H]Gal.
