JXB Advance Access published online on September 9, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern215
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RESEARCH PAPER |
The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis
Laboratoire Génome et Développement des Plantes, UMR-CNRS-IRD-Université 5096, 52 Avenue Paul Alduy, F-66860 Perpignan-cedex, France
To whom correspondence should be addressed: E-mail: devic{at}univ-perp.fr
Received 20 May 2008; Revised 28 July 2008 Accepted 29 July 2008
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Abstract |
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Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.
Key words: Embryogenesis, fertilization, N-acetyl-glucosamine, reproduction, Sanfilippo syndrome
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Introduction |
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The life cycle of flowering plants consists of a sequence of a diploid sporophytic phase and two distinct haploid gametophytic phases. Although the general pattern leading to gamete formation and the fertilization event is clear, little is known about the molecular mechanisms that regulate the transition from gametophytic to sporophytic development. The steps of these complex developmental processes seem to be dependent on an intricate network of signalling events, largely undefined and likely to involve molecules of different kinds (Preuss, 2002). Arabinogalactan proteins (AGPs) are massively glycosylated hydroxyproline-rich glycoproteins that are ubiquitous in plants and particularly abundant in the cell walls, plasma membranes, and extracellular secretion (Showalter, 2001). The pattern of distribution of specific AGP sugar residues during Arabidopsis anther and ovule development indicates that AGP-specific epitopes can constitute markers for certain cells or tissue types in very precise stages of sporogenesis and gametogenesis (Coimbra et al., 2007). AGPs also prevail in many stigma exudates, style transmitting tissues, and pollen itself, and are believed to provide recognition signals and directional guidance for the pollen tube (Wu et al., 2001). A functional genomic approach has demonstrated the essential role of Agp18 for the establishment of the female gametophytic phase in Arabidopsis (Acosta-Garcia et al., 2004). Agp19 has a more general role in plant development including reproduction (Yang et al., 2007).
In tissue cultures, AGPs are secreted into the medium from which they can be selectively precipitated with Yariv reagent (Kreuger and van Holst, 1996). A role for AGPs in plant development was initially proposed based upon their striking spatio-temporal localization as visualized by the use of monoclonal antibodies (Knox et al., 1989, 1991). A role for the JIM8 directed against sugar epitopes containing AGPs in cell–cell communication during somatic embryogenesis was proposed by McCabe et al. (1997). The AGPs required for carrot somatic embryogenesis are particularly those containing glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) although they contain only 0.2% of amine sugars in the form of GlcN and GlcNAc. These AGPs are extracellular matrix molecules (ECM) able to control or maintain cell fate when modified by endochitinases (van Hengel et al., 2001). Taken together, these observations underline the importance of sugars, especially GlcN and GlcNAc, in plant embryo development. Similarly, chitin oligosaccharides are thought to be possible patterning agents in zebrafish embryogenesis (Semino et al., 2000). The GlcNAc residues are the essential components of the chitin oligosaccharides inducing cell division in zebra fish embryonic lines by activation of specific kinases (Snaar-Jagalska et al., 2003). In mammals, heparan sulphate (HS) is a complex polymer containing repeating GlcA-GlcNAc disaccharides present at the cell surfaces. This molecule is responsible for many biological activities. HS serves as a co-receptor for various receptor tyrosine kinases, affects morphogen gradients, and hence development and organogenesis (Whitelock and Iozzo, 2005). Therefore, mutations in the enzymes involved in the synthesis or degradation of HS provoke dramatic consequences for the organism. HS has not been found in plants, however, a gene encoding an enzyme required for its catabolism is present in the plant genome suggesting the existence of a comparable as yet non-identified molecule in plants. N-acetyl-
-glucosaminidase (AtNAGLU) is similar to the well-characterized human gene whose loss of function results in the Sanfilippo syndrome or mucopolysaccharidosis IIIB (MPSS-IIIB), a rare genetic disease (Beesley et al., 2005). The pivotal role of HS in animal developmental biology and of GlcNAc residues in plant reproduction, prompted an investigation into the role of AtNAGLU, which was identified as an EMBRYO-DEFECTIVE (EMB) gene in our screening of Arabidopsis seed mutants.
In this paper, the phenotype of the atnaglu mutant is analysed and the precise spatio-temporal expression of AtNAGLU determined during the transition from gametophytic to sporophytic development. Our work demonstrates the essential role of AtNAGLU in early embryogenesis and identified the cells and tissues where modifications of glycan residues are probably occurring during plant reproduction.
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Materials and methods |
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Plant material
Arabidopsis thaliana cyl1 mutant line (accession number:ABM7) from the INRA Versailles collection of T-DNA transformed plants was of the Wassilewskija ecotype (Bechtold and Pelletier, 1998). The line was maintained in the heterozygous state. Seedlings were selected on Murashige and Skoog medium containing 100 mg l–1 of kanamycin and/or 30 mg l–1 hygromycin as required and transferred to soil after 2 weeks. Plants were grown under constant illumination at 21 °C.
Seed phenotypic analysis
Clearing and observation of immature seeds under Nomarski optics and cytological studies of seed sections were performed as previously described (Ronceret et al., 2005). Starch detection was performed on thin section of seeds embedded in resin with an iodine solution (2% potassium iodine, 0.2% iodine in water).
DNA extraction and genotyping
Genomic DNA was extracted according to Edwards et al. (1991). The left (LB) and right (RB) borders of the T-DNA insertion were identified by PCR walking (Devic et al., 1997). For genotyping, the wild-type copy was amplified as a 2 kb fragment using primers Prom5 (5'-GACTCGAGCATGATTCCGTAGCATCTTCTGGTTCACC-3') and gluco3 (5'-AATACCAATGGAGCCCAGCAAGC-3'). The cyl1 allele cannot be amplified under these conditions due to the large size of the T-DNA. The presence of the cyl1 allele was obtained by amplification of a 700 bp fragment with the primers RB1 (5'-CCAGACTGAATGCCCACAGGCCGTC-3') and gluco3. The presence of the complementation construction containing the full-length AtNAGLU cDNA was verified by amplification of a 1.8 kb fragment using primers Prom5 and gluco3.
Complementation of the cyl1 mutation
Due to the large size of the At5g13690 gene, the promoter and the cDNA were fused at the ATG. This construction contains most of the gene structures including the 5' UTR with the exception of the introns and the 3' UTR. AtNAGLU cDNA was amplified on first-strand cDNA from seedling mRNA using primers NAG5 (5'-AACCATGGATTCCATCAAATTGGTTTTGTTGGTT-3') and NAG33 (5'-CCTCAGATCATGGGAAGTATTTACTCAACAGATGC-3') to produce a double-stranded DNA fragment of 2.4 kb. The AtNAGLU cDNA was cloned into pPCRscript vector (Stratagene) and sequenced. A 1.5 kb genomic DNA fragment was amplified using primers Prom5 and Prom3 (5'-GGAATCCATGGTGATGCTTTTCCTCACTAGTGAG-3') and inserted into the pRTL2:GUS vector in front of the uidA gene (a gift from Dr Carrington, Oregon State University, Corvallis, USA). The cDNA was introduced into the AtNAGLU promoter::GUS vector by replacement of the uidA gene. The AtNAGLU promoter::cDNA cassette was introduced into the pBIB-Hyg binary vector (Becker, 1990). cyl1/+ plants were transformed by the floral dip method and the T1 seeds were selected on kanamycin and hygromycin medium in order to select for the cyl1 allele and the AtNAGLU promoter::cDNA construction.
Analysis of the AtNAGLU promoter activity
The cassette of the 1.5 kb AtNAGLU promoter fragment fused to GUS was introduced into the binary vector pBIB-Kan at the XbaI–XhoI sites (Becker, 1990). Eight independent Arabidopsis transformed plants were obtained. The GUS histochemical reaction was performed as described in Ronceret et al. (2005). The reciprocal crosses were performed with plants homozygous for the AtNAGLU:GUS transgene and originating from two independent T1 lines. Flower buds at stage 12 were emasculated. Flowers were pollinated manually 40 h after emasculation to allow the ovules to reach maturity. These manually pollinated flowers were subjected to GUS staining at various times after pollination. At each point of the kinetic of flower development, three flowers were incubated with X-gluc representing the observation of a minimum of 100 ovules.
In situ hybridization using the AtNAGLU probe
Fragments of AtNAGLU cDNA were amplified using first-strand cDNA from seedlings RNA as template. The use of primers NAG5 and NAG3 (5'-AAGTTAGTTCGTAAACCACCGGGTTC-3') resulted in the amplification of a fragment of 1.48 kb starting at the initiation codon. Primers NAG55 (5'-AACATATGGTCTTGTTTCAAGATAAGACCGCTG-3') and NAG33 amplified the last 660 nucleotides of the mRNA. The amplified DNA fragments were cloned into the pGEMT-Easy vector (PROMEGA) and sequenced. After in vitro transcription, the single-stranded RNA of 1480 nucleotides was hydrolysed to produce fragments of 500 nucleotides. Similar results were obtained with the probes from the plasmid with 1.45 kb or 660 bp inserts. Emasculated non-pollinated flowers and fertilized flowers were harvested individually and embedded into paraffin. Sections of 7–9 µm were subjected to the hybridization protocol. ISH was performed as previously described by Albert et al. (1997).
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Results |
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Characteristics of the cyclops1 mutant
embryo-defective (emb) mutants have been searched among the collection of T-DNA insertion lines from the INRA Versailles laboratory (Bechtold and Pelletier, 1998). Sporophytic lethal mutant lines producing homozygous seeds with embryos arrested at the zygote (zeus mutants) or 1 cell-embryo (cyclops mutants) were selected. The aim was to characterize genes essential for ovule development following fertilization (Ronceret et al., 2005, 2008). The seed phenotype of the ABM7 line fulfilled these requirements and the mutation was renamed cyclops1 (cyl1). Siliques of cyl1/+ plants contained one-quarter of defective seeds (Fig. 1A; Table 1) demonstrating the nuclear sporophytic recessive features of the cyl1 mutation. Most cyl1 seeds possessed embryos composed of one apical cell with one or two suspensor cells (Fig. 1; Table 1). Mutant seeds could be recognized without ambiguity as soon as wild-type seeds reached the 4–8-cell embryo stage (Fig. 1B, F). In most cyl1 seeds, the apical cell did not divide further during seed development (Fig. 1G–I). In rare instances, the apical and basal cells were able to divide, although at a low frequency (Table 1). The apical cell could produce up to a 4 cell embryo. The basal cell, which would form the suspensor, did not achieve its final structure of 7–9 cells. Although the development of the embryo and suspensor were severely reduced, the apical and basal cells followed an apparently normal pattern before the arrest of development. However, their respective development was no longer co-ordinated and, as a consequence, seeds containing embryos with one cell supported by a suspensor of 2–3 cells could be observed (Fig. 1K), whereas most wild-type one-cell embryos possessed a single elongated suspensor cell (Fig. 1J). The endosperm of cyl1 seeds was composed of 12–20 syncytial nuclei (Fig. 1F–H; Table 1), a number comparable to that of wild-type endosperm at the one cell stage (Boisnard-Lorig et al., 2001). The seed coat of cyl1 seeds at all stages of seed maturation resembled that of wild-type seeds at the preglobular stage. Staining the seed coats with iodine showed that the metabolic changes in starch biosynthesis did not occur in cyl1 seeds in contrast to wild-type seeds in the same siliques (Fig. 1L, M). In conclusion, the three main components of cyl1 seeds, embryo, endosperm, and testa, were arrested at the one-cell embryo stage. Since the mutation affected the early stages of seed development, the possibility of gametophytic effects on the cyl1 phenotype was carefully analysed. Microscopic observations of pollen grains stained by Alexander solution and of cleared ovules in cyl1/+ plants demonstrated that the mutation did not affect the morphology of the gametes (Fig. 1N, O). This was further supported by the results of reciprocal crosses (Table 2). The transmission of the T-DNA into the progeny was not affected when carried by the male or female mutant gametes and was comparable to the efficiency of wild-type gametes. Furthermore, the presence of one-quarter of abnormal seeds coincident with the phenotypically wild-type seeds at the preglobular stage, indicated that paternal and maternal alleles contributed equivalently to the developmental defect observed in cyl1 seeds.
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Cloning of the CYL1 gene
The progeny of cyl1/+ plants showed a 2:1 ratio of kanamycin resistance to sensitive seedlings (
2=1.20, n=1703) demonstrating the linkage between the T-DNA insertion (carrying the kanamycin-resistance gene) and the CYL1 gene. The flanking region of the left and right borders of the T-DNA insertion was determined by PCR walking (Devic et al., 1997). The T-DNA has inserted in the promoter region of At5g13680 and At5g13690, respectively, 179 bp and 152 bp in front of the initiation codon (Fig. 2). At5g13680 encodes ELO2/ABO1, a subunit of elongator, a histone acetyl transferase complex. Homozygous plants of the SALK_011529 and SALK_004690 lines have been studied in detail. Lack of ELO2 function resulted in the formation of rosettes with narrow leaves and reduced root growth due to a decrease in the rate of cell division (Nelissen et al., 2005). In parallel, the same SALK lines have been used to unravel the role of At5g13680 in modulating ABA and drought response and renamed ABO1 (Chen et al., 2006). The small size of abo1/elo2 seedlings was interpreted as enhanced ABA stomatal closure and increased ABA sensitivity inhibiting seedling growth. Independently, the same SALK lines (position of the T-DNA in Fig. 2) were analysed. Phenotypic analysis of these two independent alleles of At5g13680 showed no visible gametophytic or seed defects, ruling out the possibility of At5g13680 to be essential for seed development. The protein encoded by At5g13690 is homologous to alpha-N-acetylglucosaminidase (NAGLU). The essential function of this gene has been well documented in human (Zhao et al., 1996). Complementation tests of the cyl1 phenotype by expression of a wild-type copy of At5g13690 were performed. Three independent transgenic lines showed a clear reduction in the number of aborted seeds in the siliques due to expression of the AtNAGLU cDNA under the control of its own promoter (Table 3). In addition, plants carrying an RNAi construction directed against AtNAGLU presented a dramatic reduction in fertility and seed set (D Grimanelli et al., personal communication). Taken together, these data demonstrated that AtNAGLU is a gene essential for seed development. The seed phenotype of cyl1 suggested that the requirement for AtNAGLU function was precocious in embryogenesis, however, it cannot be ruled out that the reduced expression of ELO2/ABO1 in cyl1 did not contribute to the increase in the severity of the phenotype.
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AtNAGLU is homologous to a lysosomal enzyme involved in the degradation of N-glycans
AtNAGLU encodes a protein of 806 amino-acids, similar to the well-characterized human alpha-N-acetyl-glucosaminidase (EC 3.2.1.50 [EC] ) (Zhao et al., 1996). N-alpha-acetyl-glucosaminidase hydrolyses the terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides. In human cells, the enzyme is localized in lysosomes and is involved in the degradation of heparan sulphate. A single gene in the Arabidopsis genome encodes a homologue of this enzyme. Alignment of AtNAGLU with homologous proteins of plants, animals, and bacteria showed a high degree of similarity along the length of the protein (Fig. 3). The Arabidopsis protein shares approximately 60% of similarity and between 30–40% of identity with the human, mouse, and emu proteins. The plant proteins possess an additional sequence between 605–645 aa, whilst the bacterial proteins have a specific sequence between 360–390 aa. Surprisingly, no gene encoding an alpha-N-acetyl-glucosaminidase was found in the yeast genome or in the genome of other fungi.
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Expression of AtNAGLU during vegetative and reproductive development
Study of the expression of AtNAGLU by RT-PCR showed that the gene was expressed in leaf, root, floral stem, flower buds, flowers, and siliques at all stages of development (data not shown). This result was supported by the publicly available microarray expression data at genevestigator (https://www.genevestigator.ethz.ch/). In order to gain additional insights, the expression of the GUS reporter gene under the control of the AtNAGLU promoter was analysed. The 1.5 kb upstream region of AtNAGLU as used for the complementation experiment, was inserted in front of the uidA gene in a translational fusion. Eight independent transgenic lines were analysed. Although the eight lines gave similar results, three lines produced a stronger signal and were used to analyse in detail the transcriptional activity of AtNAGLU promoter. During vegetative development, expression of the reporter gene was weak and mainly found in the vasculature (Fig. 4A–C). GUS staining was clearly detected in the vascular tissues of the cotyledons (Fig. 4A) and the roots (Fig. 4C) and to a lesser extent in some sectors of the leaf vasculature (indicated by arrows in Fig. 4B). During male gametophyte development, the AtNAGLU promoter was active in the tapetum and in immature (Fig. 4D) and mature pollen grains (Fig. 4E). GUS expression was not detected in immature ovules (Fig. 4F) but was strongly expressed in the embryo sac of mature ovules at the time of fertilization (Fig. 4G). GUS expression was persistent in the endosperm of seeds at globular (Fig. 4H) and heart stage (Fig. 4I), but was absent in the endosperm of mature seeds (Fig. 4J). In the embryo, the expression of the reporter gene was detected throughout embryogenesis (Fig. 4J). Since the loss of AtNAGLU function resulted in an embryonic phenotype without obvious gametophytic effects, the kinetic of the AtNAGLU promoter activity was studied to determine whether fertilization per se was able to activate AtNAGLU expression in the embryo sac. The maternal and paternal expression of the AtNAGLU:GUS transgene in reciprocal crosses with wild-type plants were visualized. Mature pollen grains from AtNAGLU:GUS transgenic plants were used to pollinate emasculated wild-type flowers manually (Fig. 5A). Since the mature pollen grains strongly expressed the reporter gene, the actual time of fertilization was easily determined as 8 h following pollination (Fig. 5B). After the initial loading of the beta-glucuronidase enzyme from the pollen grain, no evidence of de novo transcription of the GUS gene from the paternal genome was observed in the ovule 30 h after manual pollination (Fig. 5C, D). This result is consistent with the delayed and lower level of expression of transgenes from the paternal genome in comparison to the maternal genome (Baroux et al., 2001). In the reciprocal cross, ovules expressed the GUS reporter gene in the embryo sac at the time of fertilization, 8 h after pollination (Fig. 5E, G) and this expression from the maternal genome was continued during early seed development (Fig. 5H). However, since GUS crystals could already be detected 4 h after pollination (Fig. 5F) when fertilization had not occurred yet (Faure et al., 2002), it was questioned whether the activation of the AtNAGLU promoter was induced by fertilization per se or was coincident with fertilization or was induced by pollination. Flowers from AtNAGLU:GUS transgenic plants were emasculated and stained 40 h (Fig. 5J) or 48 h after the removal of the anthers (Fig. 5I, K). The embryo sacs of the mature ovules showed a clear blue staining at 48 h, demonstrating that the AtNAGLU promoter was transcriptionally active at the time of fertilization but was not activated by pollination or fertilization. As a control, ovules of non-transformed plants (Fig. 4L) did not show a blue coloration of their embryo sacs.
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Since expression of AtNAGLU was strong during reproductive development, the detection of AtNAGLU mRNA by in situ hybridization (ISH) experiments was successful (Fig. 6). The accumulation of AtNAGLU mRNA followed a pattern similar to that revealed with the AtNAGLU promoter GUS fusion (Figs 4, 6). AtNAGLU mRNA was detected in the immature anthers in the tapetum and immature pollen grains (Fig. 6A) and in the mature pollen grains (Fig. 6B). In ovules and seed development, ISH allowed the precise localization of AtNAGLU mRNA in each cell of the embryo sac. A signal was visible in the central cell, egg cell, and synergides of ovules before fertilization (Fig. 6C). During early embryogenesis, AtNAGLU mRNA was detected in the embryo and suspensor cells and free endosperm nuclei, at one cell (Fig. 6E), globular (Fig. 6F, G), and heart embryo stages (Fig. 6H). This experiment confirmed that AtNAGLU mRNAs are present before fertilization in each cell of the embryo sac.
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Discussion |
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The cyl1 phenotype was characterized by an early arrest of seed development. Apparently, the mutation does not affect cell division per se since the endosperm contained by up to 20 nuclei while 1–2 cells composed the embryo. This phenotypic trait distinguished cyl1 from the previously described cyl2 and zeu1 mutants (Ronceret et al., 2005, 2008) in which the endosperm contained 1–4 nuclei only. CYL2 and ZEU1, respectively, encode a subunit of DNA polymerase epsilon and thymidylate kinase, proteins necessary for DNA replication occurring at the S-phase of the cell cycle. The molecular characterization of CYL1 corroborated a role distinct from the mechanisms of cell division. CYL1 encodes an enzyme involved in N-glycan degradation, an alpha-N-acetyl-glucosaminidase. Furthermore, the promoter of AtNAGLU did not contain cis elements known for cell-cycle controlled transcription. Studies of AtNAGLU expression and loss-of-function phenotype demonstrated its essential role in seed development.
In humans, NAGLU is a critical enzyme required for degradation of heparan sulphate in the lysosomes (Zhao et al., 1996). Heparan sulphate (HS) is a glycosaminoglycan composed mostly of a glucuronic linked to N-acetylglucosamine (GlcNAc) and is localized in the extracellular matrix (Whitelock and Iozzo, 2005), where it binds to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation, and tumour metastasis. Its deficiency causes a lysosomal disorder due to the over-accumulation of undegraded heparan sulphate in the lysosomes. Mutations in the other enzymes required to breakdown HS also lead to the same lysosomal storage disorder and this rare genetic disease has been named Sanfilippo syndrome or mucopolysaccharidosis III (MPS III). The MPS III type B form of the disease is due to loss of NAGLU function. The outcome of the disease is premature death by adolescence. HS has not been found in plants, but evidence for other types of glycosaminoglycans containing GlcNAc in the extracellular matrix of the plant cell exists. Plant glycosaminoglycans also play an important role in development processes. Arabinogalactan proteins (AGP) containing GlcNAc modified by EP3 chitinase, which hydrolyses internal links between two GlcNAc residues, are able to stimulate somatic embryogenesis (Passarinho et al., 2001). The action of these modified AGP on the re-initiation of cell division from a non-dividing protoplast population before they can re-synthesize their cell wall suggest that these AGPs are parietal proteins able to control cellular fate (Van Hengel et al., 2002). The observation that specific AGPs are important for female gametogenesis (Acosta-Garcia et al., 2004) open the possibility that the embryogenic competency can be established progressively after fertilization by modifying components already present prior to fertilization. Moreover, it has recently been shown that specific AGPs exhibit a differential localization in Arabidopsis suspensor and embryo cells (Hu et al., 2006). The site of accumulation of the NAGLU protein has been obtained in Arabidopsis. NAGLU has been found in the proteome of purified vacuoles from Arabidopsis rosette leaves (Carter et al., 2004). Therefore NAGLU is localized in the plant endosomal membrane system where it can play a role in the degradation of N-glycan residues. The phenotype of the loss of the catabolic enzyme AtNAGLU reveals the involvement of particular proteoglycans in developmental processes in seeds. The essential function of GlcNAc residues is further supported by the study of the loss of function of an enzyme involved in the modification of proteins by transfer of GlcNAc residues. By example, the disruption of SECRET AGENT (SEC) and SPINDLY (SPY) encoding N-acetyl-glucosamine transferases has shown that this activity is essential to the development of gametes and embryo (Hartweck et al., 2002).
At present, the substrate for the plant NAGLU enzyme is not known, but it is anticipated that it will be a molecule possessing signalling/regulatory properties. The determination of the precise localization of AtNAGLU transcripts is particularly valuable as it indicates the time and location of the remodelling of GlcNAc containing extracellular proteoglycans during fertilization. AtNAGLU is expressed in the mature pollen grain and embryo sac prior to fertilization. This expression of AtNAGLU preceded the first defects in cyl1 seeds observable after fertilization during early seed development. Furthermore, male or female gametophytic defects of the cyl1/+ plant were not observed. One explanation is that the mRNA may accumulate before the protein is produced or its activity is required. Post-transcriptional regulation of AtNAGLU could explain this time-lapse as could the lysosomal disorder hypothesis. In patients deficient in NAGLU activity, the disease is progressive. The deficiency in HS degradation per se is not detrimental, but the accumulation of partially degraded HS in the lysosomes will affect all lysosomal functions. Lysosomal dysfunction in plants could lead to an arrest of seed development. Interestingly and in contrast to yeast, a functional vacuolar system is essential for the plant cell as demonstrated by the embryonic lethal phenotype of loss of function of VCL1 (VACUOLELESS 1), the orthologue of the yeast Vps16 gene (Rojo et al., 2001). RT-PCR experiments and AtNAGLU promoter::GUS fusion showed that AtNAGLU was weakly expressed in most plant tissues. The GUS activity was detected in the vascular tissues of the seedlings and in the tapetum of immature anthers. This pattern of expression indicates that degradation of glycosaminoglycans also occurs during vegetative development, although the main role of AtNAGLU is taking place during reproduction.
In conclusion, the identification of AtNAGLU, an enzyme known to be involved in the degradation of extracellular proteoglycans, together with the determination of its precise expression pattern, provide novel information on the time and location of the modifications of extracellular GlcNAc residues during development and, in particular, during the fertilization process.
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Acknowledgements |
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We thank Nicole Bechtold and Roger Voisin for their work in the T-DNA insertion collection (Versailles) and Georges Pelletier for the access to the collection. We thank Thomas Roscoe for critical reading of the manuscript and help in improving the English. AR was a recipient of a French doctoral fellowship from the University of Perpignan.
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Footnotes |
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* Present address: Plant Breeding and Genetics, 418 Bradfield Hall, Cornell University, Ithaca, NY14850, USA.
Present address: Genomics Laboratory, Institute for Molecular and Cellular Biology of Plants, UPV-CSIC Av. Naranjos s/n, E-46022 Valencia, Spain.
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Abbreviations |
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emb, embryo-defective; NAGLU, alpha-N-acetyl-glucosaminidase; GlcNAc, N-acetyl-glucosamine.
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References |
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