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• W1983338961 abstract "d-Apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of d-apiose involves UDP-d-apiose/UDP-d-xylose synthase catalyzing the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose. In this study we have analyzed the cellular effects of depletion of UDP-d-apiose/UDP-d-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-d-apiose/UDP-d-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of d-apiose as well as 2-O-methyl-l-fucose and 2-O-methyl-d-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a d-apiose deficiency. d-Apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of d-apiose involves UDP-d-apiose/UDP-d-xylose synthase catalyzing the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose. In this study we have analyzed the cellular effects of depletion of UDP-d-apiose/UDP-d-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-d-apiose/UDP-d-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of d-apiose as well as 2-O-methyl-l-fucose and 2-O-methyl-d-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a d-apiose deficiency. The primary wall of higher plants consists of a rigid cellulose-xyloglucan network that is embedded in and interacts with a pectin network. Rhamnogalacturonan II (RG-II) 4The abbreviations used are: RG-II, rhamnogalacturonan II; RT, reverse transcription; kb, kilobase; GFP, green fluorescent protein; VIGS, virus-induced gene silencing; TRV, tobacco rattle virus; H2DCFDA, 2,7-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; HR, hypersensitive response. 4The abbreviations used are: RG-II, rhamnogalacturonan II; RT, reverse transcription; kb, kilobase; GFP, green fluorescent protein; VIGS, virus-induced gene silencing; TRV, tobacco rattle virus; H2DCFDA, 2,7-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; HR, hypersensitive response. is a structurally complex pectic polysaccharide found in primary walls of angiosperms and gymnosperms. RG-II is composed of at least 12 different glycosyl residues linked together by more than 20 different glycosidic linkages, but despite this complexity, the glycosyl-residue composition of RG-II is remarkably conserved among species (1Pérez S. Rodríguez-Carvajal M.A. Doco T. Biochimie (Paris). 2003; 85: 109-121Crossref PubMed Scopus (208) Google Scholar, 2O'Neill M.A. Ishii T. Albersheim P. Darvill A.G. Annu. Rev. Plant Biol. 2004; 55: 109-139Crossref PubMed Scopus (677) Google Scholar). RG-II exists predominantly as a dimer that is covalently cross-linked by a borate diester. Borate cross-linking of RG-II additionally results in cross-linking of the two homogalacturonan chains upon which the RG-II molecules are constructed (2O'Neill M.A. Ishii T. Albersheim P. Darvill A.G. Annu. Rev. Plant Biol. 2004; 55: 109-139Crossref PubMed Scopus (677) Google Scholar). These processes of cross-linking are thought to result in a stable three-dimensional pectin network. Although little is known about how the structural complexity of RG-II contributes to its biological function, evidence suggests that the structural integrity of the RG-II molecule is essential for normal borate cross-link formation. The mur1 mutant is dwarfed and has brittle stems (3Reiter W.-D. Chapple C.C.S. Somerville C.R. Science. 1993; 261: 1032-1035Crossref PubMed Scopus (214) Google Scholar), and these defects are caused by a mutation in GMD2 encoding a GDP-mannose 4,6-dehydratase, an enzyme required for the biosynthesis of l-fucose (4Bonin C.P. Potter I. Vanzin G.F. Reiter W.-D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2085-2090Crossref PubMed Scopus (190) Google Scholar). RG-II synthesized by the mur1 mutant completely lacks l-Fuc residues, and only ∼50% of the RG-II is cross-linked by borate (5O'Neill M.A. Eberhard S. Albersheim P. Darvill A.G. Science. 2001; 294: 846-849Crossref PubMed Scopus (502) Google Scholar). Interestingly, the mur2 mutant, which is defective in a xyloglucan-specific fucosyltransferase, synthesizes xyloglucan that contains little l-Fuc but grows normally (6Vanzin G.F. Madson M. Carpita N.C. Raikhel N.V. Keegstra K. Reiter W.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3340-3345Crossref PubMed Scopus (211) Google Scholar), indicating that xyloglucan fucosylation is not critical for plant growth regulation and wall strength. Taken together, these results suggest that the dwarfed phenotype of mur1 plants is caused by reduced cross-linking of RG-II due to lack of l-fucose residues. Supporting this, an exogenous supply of borate rescues the growth defect of the mur1 mutant (5O'Neill M.A. Eberhard S. Albersheim P. Darvill A.G. Science. 2001; 294: 846-849Crossref PubMed Scopus (502) Google Scholar). The nolac-H18 tobacco callus mutant shows reduced cell adhesion and defective shoot development, which is caused by altered RG-II structure (7Iwai H. Masaoka N. Ishii T. Satoh S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16319-16324Crossref PubMed Scopus (203) Google Scholar). The RG-II of the mutant lacks the α-l-Galp-(1 → 2)-β-d-GlcpA-(1 → portion of side chain A. Only ∼50% of the RG-II in the mutant walls is present as a dimer compared with >95% dimer formation in wild-type walls. Expression of NpGUT1 encoding a putative glucuronosyltransferase in the mutant rescues the mutant phenotype, including restoration of borate cross-linking to normal levels (7Iwai H. Masaoka N. Ishii T. Satoh S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16319-16324Crossref PubMed Scopus (203) Google Scholar). These results suggest that the α-l-Galp-(1 → 2)-β-d-GlcpA-(1 → portion of side chain A is required for normal borate cross-linking. Finally, the bor1 mutant has a defective efflux type transporter that mediates borate transfer (8Takano J. Noguchi K. Yasumori M. Kobayashi M. Gajdos Z. Miwa K. Hayashi H. Yoneyama T. Fujiwara T. Nature. 2002; 420: 337-340Crossref PubMed Scopus (468) Google Scholar). The wild-type and bor1 RG-II have similar glycosyl residue compositions, but ∼60% of RG-II in bor1 cell walls is present as the monomer (9Noguchi K. Ishii T. Matsunaga T. Kakegawa K. Hayashi H. Fujiwara T. J. Plant Nutr. Soil Sci. 2003; 166: 175-178Crossref Scopus (25) Google Scholar). Borate treatment rescues the dwarfism and the extent of RG-II cross-linking of the bor1 mutant. Thus, reduced cross-linking of RG-II is likely responsible for the dwarfed phenotype of the bor1 mutant. d-Apiose is only present in RG-II in the cell walls of most higher plants, and it serves as an attachment point for two highly complex side chains to the homogalacturonan backbone (10Stevenson T.T. Darvill A.G. Albersheim P. Carbohydr. Res. 1988; 182: 207-226Crossref Scopus (62) Google Scholar). The two vicinal hydroxyl groups in the furanose ring of d-apiose form a cyclic diester with borate to result in RG-II cross-linking. Biosynthesis of d-apiose has been mostly studied in the duckweed Lemna minor and in parsley, where d-apiose is an abundant component of apiogalacturonan and apiin, respectively (11Golovchenko V.V. Ovodova R.G. Shashkov A.S. Ovodov Y.S. Phytochemistry. 2002; 60: 89-97Crossref PubMed Scopus (67) Google Scholar, 12Hahlbrock K. Knobloch K.H. Kreuzaler F. Potts J.R. Wellmann E. Eur. J. Biochem. 1976; 61: 199-206Crossref PubMed Scopus (140) Google Scholar). In both duckweed and parsley, highly purified fractions of UDP-d-apiose synthase activity still contained UDP-d-xylose synthase activity, indicating that both reactions are catalyzed by a single enzyme, termed UDP-d-apiose/UDP-d-xylose synthase. Recently, Mølhøj et al. (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar) cloned the AXS1 gene encoding this synthase from Arabidopsis and demonstrated that the recombinant AXS1 protein indeed has both UDP-d-apiose synthase and UDP-d-xylose synthase activities. The Arabidopsis genome has a second gene (AXS2) that encodes a protein with 96% sequence identity to AXS1, and both genes are expressed in all Arabidopsis tissues. These results indicate that Arabidopsis has at least two UDP-d-apiose/UDP-d-xylose synthases. Consequently, an Arabidopsis line carrying a T-DNA within the AXS1 gene did not show any visible defects. 5R. Verma and W.-D. Reiter, unpublished results. 5R. Verma and W.-D. Reiter, unpublished results. In this study we show that depletion of UDP-d-apiose/UDP-d-xylose synthases results in RG-II deficiency in the cell walls by using VIGS of a Nicotiana benthamiana homolog of the AXS genes, designated NbAXS1. Cell wall abnormalities including incomplete walls and wall thickening were correlated with reduced levels of the RG-II-specific monosaccharides d-apiose, 2-O-methyl-l-fucose, and 2-O-methyl-d-xylose. Thus, the apparent dwarfism and cell death of the NbAXS1 VIGS plants were likely caused by RG-II deficiency due to a defect in the biosynthesis of UDP-d-apiose. Virus-induced Gene Silencing—N. benthamiana plants were grown in a growth room at 24 °C under a regime of 16 h of light and 8 h of dark. cDNA segments from NbAXS1 were PCR-amplified and cloned into the pTV00 vector containing a part of the tobacco rattle virus (TRV) genome (14Ratcliff F. Martin-Hernansez A.M. Baulcombe D.C. Plant J. 2001; 25: 237-245Crossref PubMed Scopus (727) Google Scholar) using BamHI and ApaI sites. Virus-induced gene silencing was carried out as described (15Kim M. Ahn J.-W. Jin O. Paek K.-H. Pai H.-S. J. Biol. Chem. 2003; 278: 19406-19415Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). For RT-PCR analyses, the fourth leaf above the infiltrated leaf was used. DNA and RNA Gel Blot Analysis—For DNA gel blot analysis, the genomic DNA isolated from leaves of N. benthamiana was digested with EcoRI, HindIII, and EcoRV, electrophoresed on a 0.8% agarose gel, and blotted onto Hybond-N nylon membrane (Amersham Biosciences). Prehybridization and hybridization was carried out as described (16Lee S.S. Cho H.S. Yoon G.M. Ahn J.-W. Kim H.H. Pai H.-S. Plant J. 2003; 33: 825-840Crossref PubMed Scopus (106) Google Scholar). For RNA gel blot analysis using plant tissues, total RNA was prepared by using TRIzol™ reagent (Invitrogen) following the manufacturer's instructions. Approximately 30 μg of total RNA was electrophoresed on an agarose gel containing 5.1% (w/v) formaldehyde and blotted onto Hybond-N nylon membrane. Prehybridization and hybridization was carried out as described (16Lee S.S. Cho H.S. Yoon G.M. Ahn J.-W. Kim H.H. Pai H.-S. Plant J. 2003; 33: 825-840Crossref PubMed Scopus (106) Google Scholar). For DNA and RNA gel blot analyses, the probe was the 0.6-kb PCR fragment corresponding to the C-terminal region of the NbAXS1 cDNA. Semiquantitative RT-PCR—Semiquantitative RT-PCR was carried out using 5 μg of total RNA as described (16Lee S.S. Cho H.S. Yoon G.M. Ahn J.-W. Kim H.H. Pai H.-S. Plant J. 2003; 33: 825-840Crossref PubMed Scopus (106) Google Scholar). Primers for RT-PCR were made according to published cDNA sequences as described (15Kim M. Ahn J.-W. Jin O. Paek K.-H. Pai H.-S. J. Biol. Chem. 2003; 278: 19406-19415Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Subcellular Localization of the NbAXS1 Protein—The NbAXS1 cDNA corresponding to the entire coding region was cloned into the 326-GFP plasmid (17Lee Y.J. Kim D.H. Kim Y.-W. Hwang I. Plant Cell. 2001; 13: 2175-2190Crossref PubMed Scopus (183) Google Scholar) using XbaI sites to generate the NbAXS1-GFP fusion protein. The fusion constructs were introduced into Arabidopsis protoplasts prepared from seedlings by polyethylene glycol-mediated transformation (18Abel S. Theologis A. Methods Mol. Biol. 1998; 82: 209-217PubMed Google Scholar). Expression of the fusion constructs was monitored by a confocal laser scanning microscope (Carl Zeiss LSM 510) at 24 h after transformation. The filter sets were BP505-530 (excitation 488 nm, emission 505-530 nm) and LP650 (excitation 488 nm, emission 650 nm) (Carl Zeiss) for green fluorescent protein and autofluorescence of chlorophyll, respectively. Western Blot Analysis of NbAXS1-GFP—GFP or NbAXS1-GFP fusion constructs were introduced into Arabidopsis protoplasts by polyethylene glycol-mediated transformation. At 24 h after transformation proteins were extracted from the protoplasts. As a control, proteins from untreated protoplasts were also prepared. Western blot analysis was carried out as described (16Lee S.S. Cho H.S. Yoon G.M. Ahn J.-W. Kim H.H. Pai H.-S. Plant J. 2003; 33: 825-840Crossref PubMed Scopus (106) Google Scholar). Thirty μg of proteins were electrophoresed on a 10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and probed with a polyclonal antibody raised against green fluorescent protein (GFP) (1:2000 dilution; Clontech). They were then reacted with anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:5000 dilution; Amersham Biosciences) and with ECL reagent (Amersham Biosciences) for detection. Measurement of the Mitochondrial Membrane Potential—Tetramethylrhodamine methyl ester (Molecular Probes) was added to protoplasts isolated from leaves of the VIGS lines at a final concentration of 200 nm. After incubation for 1-2 min at 37 °C, the protoplasts were transferred to wells on microscope slides and examined under a confocal microscope (Carl Zeiss LSM 510) with optical filters (543-nm excitation, 585-nm emission) to visualize the red fluorescent probe. Quantitative images were captured, and the data were analyzed using the LSM 510 program (Version 2.8). Measurement of H2O2 Production in Vivo—Protoplasts isolated from leaves of the VIGS lines were incubated in 2 mm 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes) for 30, 60, 90, 120, and 150 s. Protoplasts were transferred to wells on microscope slides and examined under a confocal microscope (Carl Zeiss LSM 510) with optical filters (488 nm excitation, 505 nm emission) to visualize the oxidized green fluorescent probes. Measurement of Ion Leakage and Chlorophyll Contents, Detection of Autofluorescence, and Evans Blue Staining—After Agrobacterium infiltration, the fourth leaf above the infiltrated leaf from the TRV control and TRV:NbAXS1 lines was collected and analyzed. Measurement of membrane leakage, detection of autofluorescence, and Evans blue staining were performed as described (15Kim M. Ahn J.-W. Jin O. Paek K.-H. Pai H.-S. J. Biol. Chem. 2003; 278: 19406-19415Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Total chlorophyll contents were measured as described (19Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4660) Google Scholar). Analysis of Starch Content—Leaves were harvested from the VIGS lines at 20 days after infiltration and bleached in 80% (v/v) ethanol. After rinsing with double-distilled water, the leaves were stained with Lugol's iodine staining reagent (Sigma) and briefly destained with water. Measurement of Peroxidase and Ascorbate Peroxidase Activity—Peroxidase and ascorbate peroxidase activities were measured as described (15Kim M. Ahn J.-W. Jin O. Paek K.-H. Pai H.-S. J. Biol. Chem. 2003; 278: 19406-19415Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Electron Microscopy—The fourth leaf above the infiltrated leaf from the TRV and TRV:NbAXS1 lines was fixed, processed, and embedded in Spurrs epoxy resin as described (20Lee H.-S. Karunanandaa B. McCubbin A. Gilroy S. Kao T.-H. Plant J. 1996; 9: 613-624Crossref Scopus (67) Google Scholar). Leaves were sectioned (1-μm-thick sections) with an ultramicrotome (model MT-600; Sorvall) and stained in 1% toluidine blue. Ultrathin leaf sections were then generated and placed on Formvar-coated copper grids, where they were stained with lead citrate and uranyl acetate. The sections were observed in a JEOL electron microscope (model 100CX II) at 80 kV, and pictures were taken using Kodak Electron Microscope film no. 4489. Production of the Recombinant NbAXS1 Protein and Measurement of Its Enzyme Activity—The coding region of the NbAXS1 cDNA was PCR-amplified and cloned into the pET29a vector (Invitrogen) using NdeI and XhoI sites. Conditions for the induction of gene expression and the purification of recombinant NbAXS1 and AXS1 via nickel nitrilotriacetic acid affinity chromatography were as described (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar). Assays for UDP-d-apiose/UDP-d-xylose synthase activity were conducted with 3 μg of the purified recombinant protein, and the formation of products was monitored by gas liquid chromatography of alditol acetates as described (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar). Monosaccharide Composition Analysis of the Cell Walls—A polysaccharide fraction containing predominantly cell wall material was prepared from the leaves of the VIGS lines by repeated extractions with 70% (v/v) ethanol as described (3Reiter W.-D. Chapple C.C.S. Somerville C.R. Science. 1993; 261: 1032-1035Crossref PubMed Scopus (214) Google Scholar). The polysaccharide was hydrolyzed with sulfuric acid, and the monosaccharide composition was determined as alditol acetates by gas-liquid chromatography as described (3Reiter W.-D. Chapple C.C.S. Somerville C.R. Science. 1993; 261: 1032-1035Crossref PubMed Scopus (214) Google Scholar). Because variable amounts of starch were found in the polysaccharide fraction, glucose was omitted from the calculation of the relative amounts of monosaccharide components. Virus-induced Gene Silencing of NbAXS1 Putatively Encoding UDP-d-apiose/UDP-d-xylose Synthase—Previously, we used TRV-based VIGS in N. benthamiana to assess the functions of various signaling genes and genes whose suppressed expression may be lethal in embryos or seedlings (15Kim M. Ahn J.-W. Jin O. Paek K.-H. Pai H.-S. J. Biol. Chem. 2003; 278: 19406-19415Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 21Ahn J.-W. Kim M. Lim J.H. Kim G.-T. Pai H.-S. Plant J. 2004; 38: 969-981Crossref PubMed Scopus (65) Google Scholar, 22Park J.-A. Ahn J.-W. Kim Y.-K. Kim S.J. Kim J.-G. Kim W.T. Pai H.-S. Plant J. 2005; 42: 153-163Crossref PubMed Scopus (100) Google Scholar). Approximately 15,000 ESTs were sequenced from three cDNA libraries constructed from various tissues of N. benthamiana, and >2,000 selected cDNAs were subjected to VIGS. The screening revealed that gene silencing of a homolog of Arabidopsis AXS genes encoding UDP-d-apiose/UDP-d-xylose synthase (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar) severely inhibited plant growth and caused premature leaf yellowing. The screening vector contained the full-length cDNA, which was designated NbAXS1. The NbAXS1 cDNA is 1656 bp in length and encodes a polypeptide of 387 amino acids corresponding to a molecular mass of 43,345 Da. The amino acid sequence of NbAXS1 was aligned with closely related sequences from Arabidopsis, tomato, potato, and rice (supplemental Fig. 1). NbAXS1 showed an 87-88% amino acid sequence identity to Arabidopsis AXS1 and AXS2 (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar) and 94, 93, and 83% sequence identity to homologs of potato, tomato, and rice, respectively. Genomic Organization and Expression of NbAXS1—DNA gel blot analysis was performed with the genomic DNA from N. benthamiana digested with restriction enzymes (supplemental Fig. 2A). The probe was the 0.6-kb PCR fragment corresponding to the C-terminal region of the NbAXS1 cDNA. EcoRI digestion resulted in four hybridizing bands, whereas HindIII and EcoRV digestion showed three hybridizing bands. These results suggest that the N. benthamiana genome contains at least two NbAXS1-related genes. This result and our previous finding that the Arabidopsis T-DNA knock-out mutant of the AXS1 gene did not show any visible defects indicate that the VIGS phenotype observed in this study is likely to be caused by gene silencing of multiple NbAXS1-homologous genes. We examined expression of the NbAXS1 mRNAs in different tissues of N. benthamiana plants using RNA gel blot analysis. The ∼1.9-kb NbAXS1 transcripts were detected in roots, stems, leaves, and flowers at similar levels (supplemental Fig. 2B). UDP-d-apiose/UDP-d-xylose Synthase Activity of the Recombinant NbAXS1 Protein—To confirm that NbAXS1 indeed encodes a UDP-d-apiose/UDP-d-xylose synthase in N. benthamiana, the enzymatic activity of the recombinant NbAXS1 protein was examined in comparison with that of AXS1 from Arabidopsis (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar). The coding region of the full-length NbAXS1 was cloned into the pET29a expression vector and expressed in Escherichia coli. The recombinant NbAXS1 protein was affinity-purified using the C-terminal histidine tag, yielding a polypeptide ∼44 kDa in size, which is consistent with the predicted size of NbAXS1 (data not shown). After incubation of the recombinant NbAXS1 and AXS1 proteins with UDP-d-glucuronate, the reaction products were hydrolyzed, and the resulting monosaccharides were separated and quantified by gas liquid chromatography of alditol acetates. NbAXS1 converted UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose as did AXS1 (Fig. 1A). In contrast, heat-inactivated NbAXS1 did not catalyze the reaction (Fig. 1A). These results demonstrate that NbAXS1 encodes a functional UDP-d-apiose/UDP-d-xylose synthase. A comparison of the kinetic parameters of NbAXS1 and AXS1 from Arabidopsis (13Mølhøj M. Verma R. Reiter W.-D. Plant J. 2003; 35: 693-703Crossref PubMed Scopus (69) Google Scholar) did not reveal any significant differences between the two enzymes. Similarly, the relative amounts of UDP-d-apiose and UDP-d-xylose produced by NbAXS1 and AXS1 were essentially the same (Fig. 1A). Subcellular Localization of the NbAXS1-GFP Fusion Protein—Subcellular distribution of the NbAXS1 protein in plant cells was examined by expressing a fusion protein between NbAXS1 and GFP. DNA constructs encoding NbAXS1-GFP or GFP alone under the control of the CaMV35S promoter were introduced into protoplasts isolated from Arabidopsis seedlings. After incubation at 25 °C, expression of the introduced genes was examined under a confocal laser scanning microscope with different filters to capture the image of GFP and autofluorescence of chlorophyll. After 24 h of incubation, the green fluorescent signal of the NbAXS1-GFP fusion protein was mostly localized in the cytosol, similar to the localization of a GFP control (Fig. 1B). To examine if the NbAXS1-GFP fusion protein is expressed intact in the protoplasts, the fusion protein was detected by using Western blotting (Fig. 1C). The NbAXS1-GFP fusion construct or the GFP vector alone were transformed into Arabidopsis protoplasts. As a control, the protoplasts were mock-transformed. After 24 h of incubation, total proteins were prepared and fractionated on SDS-PAGE. Western blot analysis was performed using the anti-GFP polyclonal antibody to detect GFP or NbAXS1-GFP fusion protein. The antibody visualized a ∼30-kDa protein band in the GFP-transformed fraction and a ∼75-kDa band in the NbAXS1-GFP-transformed fraction but none in the mock-transformed fraction. The size of the protein bands is consistent with the expected size of the GFP and the NbAXS1-GFP proteins, indicating that the green fluorescent signal detected in the NbAXS1-GFP-transformed protoplasts accurately depicts the subcellular localization of NbAXS1-GFP. VIGS Phenotypes and Suppression of the Endogenous NbAXS1 Transcripts—To confirm gene silencing of NbAXS1, four different fragments of the NbAXS1 cDNA were cloned into the TRV-based VIGS vector pTV00 (14Ratcliff F. Martin-Hernansez A.M. Baulcombe D.C. Plant J. 2001; 25: 237-245Crossref PubMed Scopus (727) Google Scholar), and N. benthamiana plants were infiltrated with Agrobacterium containing each plasmid (Fig. 2A). TRV:NbAXS1(N) and TRV:NbAXS1(C) contained a 0.6-kb N- and 0.6-kb C-terminal half of the coding region, respectively, whereas TRV:NbAXS1(F) contained a full-length NbAXS1-coding region. TRV:NbAXS1(T) contained a 90-bp cDNA fragment encoding the C-terminal end of the coding region. VIGS with all four constructs resulted in the same phenotype of growth arrest and senescence-like leaf yellowing (Fig. 2B). Newly emerged leaves were small and yellowish and made a leaf cluster near the shoot apex, and the stem growth was severely inhibited. The effects of gene silencing on the endogenous amounts of the NbAXS1 mRNA were examined by semiquantitative RT-PCR (Fig. 2C). Primers for RT-PCR were designed to exclude the cDNA regions used in the VIGS constructs, and the transcript level for actin was measured as a control. RT-PCR using NbAXS1-N primers (Fig. 2A) that detect the N-terminal region of the NbAXS1 cDNA produced significantly reduced amounts of PCR products in the VIGS lines of TRV:NbAXS1(C) and TRV:NbAXS1(T) compared with the TRV control, indicating that the endogenous level of the NbAXS1 transcripts was greatly reduced in those plants. The same primers detected high levels of viral genomic transcripts containing the N-terminal region of NbAXS1 in the TRV:NbAXS1(N) and TRV:NbAXS1(F) lines. In contrast, NbAXS1-C primers (Fig. 2A) that recognize the C-terminal region of the cDNA showed suppression of the endogenous NbAXS1 transcripts in the TRV:NbAXS1(N) and TRV: NbAXS1(T) lines, whereas they detected the viral genomic transcripts in the TRV:NbAXS1(C) and TRV:NbAXS1(F) lines. The transcript levels of actin remained constant. These results demonstrate that expression of NbAXS1 was significantly reduced in the VIGS lines. Cell Wall Defects and Premature Cell Death—We examined cell morphology of the leaves from the TRV:NbAXS1 VIGS lines using light and transmission electron microscopy compared with a TRV control. Transverse leaf sections revealed that the TRV control leaves had the typical leaf structure of dicotyledonous plants with distinct adaxial and abaxial epidermal layers (Fig. 3, A and C). TRV:NbAXS1 lines showed swollen cells filled with dense granule-like structures (Fig. 3, B and D). Strikingly, some cells exhibited wall gaps (marked by the black arrows in Fig. 3, B and D), and a few seemed to be disintegrating with ruptured cell walls (marked by the white arrow in Fig. 3D). Despite these abnormalities, the number of cell layers and the typical dorsoventral organization of the palisade and mesophyll cells were mostly maintained in the TRV: NbAXS1 lines. The incomplete cell walls and cell rupturing observed in TRV:NbAXS1 lines indicate that depletion of NbAXS1 causes abnormal cell wall biogenesis. Transmission electron microscopic analysis of the leaf cells of the TRV: NbAXS1 lines showed dramatic degeneration of organelles and cellular ultrastructure (Fig. 3F, cf. control in Fig. 3E). The cells contained reduced amounts of cytosol, apparently no or an abnormal nucleus, and reduced numbers of chloroplasts. Some of the chloroplasts were undergoing degradation (marked by the arrows in Fig. 3F). The cells accumulated a large number of electron-dense particles that are likely the remains of disintegrating chloroplasts and other cellular compartments (Fig. 3F). Accumulation of Cell Death-related Markers and Excessive Reactive Oxygen Species (ROS) Production—The physiology of the cell death phenotype in NbAXS1 VIGS lines was examined. We stained the detached leaves of the VIGS lines with Evans blue, a dye that is excluded by the membranes of living cells but diffuses into dead cells. The TRV: NbAXS1 lines exhibited black staining in" @default.
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• W1983338961 title "Depletion of UDP-d-apiose/UDP-d-xylose Synthases Results in Rhamnogalacturonan-II Deficiency, Cell Wall Thickening, and Cell Death in Higher Plants" @default.
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