JXB Advance Access originally published online on April 10, 2007
Journal of Experimental Botany 2007 58(7):1813-1823; doi:10.1093/jxb/erm040
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
The ectopically parting cells 1-2 (epc1-2) mutant exhibits an exaggerated response to abscisic acid
1Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK
2Department of Biochemistry, Downing site, Cambridge CB2 1QW, UK
3Forestry and Forest Products Research Institute, 1 Matunosato, Tsukuba, Ibaraki, 305-8687, Japan
To whom correspondence should be addressed. E-mail: stephen.jackson{at}warwick.ac.uk
Received 6 December 2006; Revised 14 February 2007 Accepted 14 February 2007
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Abstract |
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The ECTOPICALLY PARTING CELLS 1 (EPC1) gene encodes a putative retaining glycosyltransferase of the GT64 family, and epc1-1 mutant plants have a severely dwarfed phenotype. A new mutant allele of this gene, epc1-2, has been isolated. Reduced cell adhesion that has previously been reported for the epc1-1 mutant was not observed for either the epc1-1 or epc1-2 mutants grown in our conditions, suggesting that EPC1 does not affect cell adhesion but is involved in some other process affecting plant growth and development. It is shown that the epc1-2 mutant exhibits hypersensitivity to the phytohormone abscisic acid in germination and root elongation assays, however it shows an unaltered response to gibberellin, epi-brassinosteroid, auxin, or ethylene. An EPC1:YFP fusion protein is localized to small motile structures within the cytosol that are similar in size and number to the Golgi apparatus. Analysis of cell wall pectins revealed that levels of ß-(1,4)-galactan in the epc1-2 mutant are reduced by 50%, whilst other pectic polysaccharides (homogalacturonan, arabinan, and rhamnogalacturonan II) are unchanged.
Key words: ABA, cell wall, dwarf, galactan, pectin, root hair
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Introduction |
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Glycosyltransferases are a diverse class of enzymes that catalyse the biosynthesis of polysaccharides and the glycosylation of proteins and small molecules such as plant hormones (Zhong and Ye, 2003). Glycosyltransferases have been classified into at least 65 families based upon amino acid sequence similarities (Coutinho et al., 2003; http://afmb.cnrs-mrs.fr/CAZY/). 408 putative glycosyltransferases (35 families) have been identified in Arabidopsis, however the biochemical activities and biological functions of most glycosyltransferases in Arabidopsis have yet to be characterized.
The isolation of the ectopically parting cells 1–1 (epc1-1) mutant which is mutated in a gene for a putative glycosyltransferase of the GT64 family was described by Singh et al. (2005). The EPC1 gene encodes a hypothetical protein with 33% amino acid identity (50% similarity) to the C-terminal half of mammalian multiple exostoses proteins 1 and 2 (EXT 1 and EXT 2), and the Drosophila TOUT-VELU (TTV) protein (Bellaiche et al., 1998; Lind et al., 1998). The C-terminal domains of these proteins contain the N-acetyl-D-glucosaminyl (GlcNAc) transferase activity (Senay et al., 2000), which is a GT64 glycosyltransferase and which catalyses the addition of 
-(1,4)-GlcNAc residues. The GT64 family of glycosyltransferases are retaining enzymes and so result in a product that has the same anomeric configuration as the nucleotide sugar substrate.
The EPC1 protein contains a conserved DXD motif common to most glycosyltransferases that is postulated to be involved in the co-ordination of a divalent cation at the nucleotide sugar binding site (Wiggins and Munro, 1998; Fig. 1). Both the EXT and TTV proteins contain a single membrane spanning domain near the N-terminus and a predicted membrane spanning domain is also present in a similar location in EPC1 (Fig. 1). The short N-terminal domain followed by a single transmembrane domain and a large C-terminal catalytic domain is the common topology of type II membrane Golgi-localized glycosyltransferases (Paulson and Colley, 1989).
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The epc1-1 mutant exhibits a severe dwarf phenotype and is reported to have reduced cellular adhesion which was postulated to be the cause of the growth phenotype. In this paper the characterization of another mutant allele of EPC1 called epc1-2 is described. Reduced cell adhesion was not observed in either the epc1-2 or epc1-1 mutant in our conditions, raising the possibility that this gene is not involved in cell adhesion but plays some other role in plant growth and development. It is shown that the epc1-2 mutant exhibits an exaggerated response to exogenous abscisic acid (ABA), but not to any other hormone tested. Evidence is also presented that supports the prediction that EPC1 is localized to the Golgi apparatus. Together these observations raise the possibility that one role of the EPC1 glycosyltransferase may be to glycosylate a component(s) located in or outside the plasma membrane which negatively affects the response to ABA.
Many components involved in ABA signal transduction have still to be isolated, although the identification of FCA and the Mg-chelatase H subunit as ABA receptors, together with the characterization of mutants with altered responses to ABA, has led to substantial progress in recent years (Finkelstein et al., 2002; Razem et al., 2006; Shen et al., 2006). Many genes involved in ABA signalling have been shown to encode negative regulators of ABA sensitivity, for example, ABI1, ABI2, ERA1, and ROP10 (Gosti et al., 1999; Merlot et al., 2001; Yang, 2002; Zheng et al., 2002). Localization to the plasma membrane is essential for ROP10 function (Zheng et al., 2002), and both membrane-localized heterotrimeric G proteins and a plasma membrane leucine-rich repeat receptor kinase have been shown to be involved in ABA signalling (Pandey and Assmann, 2004; Osakabe et al., 2005). To date, the majority of proteins that have been identified as potential regulators of ABA are located in the plasma membrane or in the cytosol and little is known about possible roles of extracellular components that might affect the response to ABA at the plasma membrane. There is, however, a report of an extracellular arabinogalactan protein (AGP), AtAGP30, which is involved in ABA perception at the root tip (van Hengel and Roberts, 2003). The characterization of such extracellular factors and their biosynthesis would greatly enhance our understanding of the mechanisms of ABA signalling.
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Materials and methods |
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Materials
Seeds were obtained from the Nottingham Arabidopsis Stock Centre. GalU, (GalU)3, endo-PG (EC 3.2.1.15 [EC] ) from Aspergillus japonicus, Gal, Ara, urea, IAA, NAA, ACC, and ABA were purchased from Sigma (Poole, Dorset, UK). H Gilbert (University of Newcastle, UK) kindly provided
-(1,5)-arabinanase (Pseudomonas fluorescens) and ß-(1,4)-galactanase (P. fluorescens). All enzymes used in this analysis were analysed for the purity or/and specificity using the PACE method as described in Goubet et al. (2002). All were specific and pure (data not shown). 8-Amino naphthalene-1,3,6-trisulphonic acid (ANTS) and 2-aminoacridone (AMAC) were purchased from Molecular Probes (Leiden, The Netherlands). The mouse monoclonal antibody CCRC-M7 was kindly provided by M Hahn (CCRC, University of Georgia, USA). The rat monoclonal antibody JIM13 was kindly provided by P Knox (University of Leeds, UK). Secondary antibodies (Cy3-conjugated goat anti-rabbit, anti-rat, or anti-mouse) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Plant growth conditions
Prior to growth on agar, seeds were sterilized for 10 min in 30% bleach, then washed several times with sterile water to remove residual bleach. Sterile seeds were plated on Murashige and Skoog (MS) plus agar growth media (Duchefa) supplemented with 2% sucrose and hormones where indicated. Plants both in media and in soil were grown in 16/8 h light/dark in Sanyo MLR-350 growth cabinets (Sanyo Gallenkamp plc) at 22 °C and 70% humidity under fluorescent lights at 100 µmol m–2 s–1. PPT was sprayed on 2-week-old soil-grown plants at a concentration of 100 mg ml–1.
Plants for cell wall and immunolocalization experiments were grown on soil in 10/14 h light/dark, with 65% humidity. Leaves from non-flowering plants were collected to make cell walls. For immunolocalization, hypocotyl, leaf, and the lower and upper parts of the stem were taken from 6-week-old plants. As epc1-2 flowered slightly later than WT and epc1-2–C1 plants, the epc1-2 samples for these analyses were taken 10 d later.
Inverse PCR
600 ng epc1-2 genomic DNA was digested with SpeI and self-ligated overnight at 16 °C.
Inverse-PCR was done using 100 ng self-ligated DNA and primers to the T-DNA insertion RB-K1; 5'-CGAAACGCAGCACGATACG-3'; LB-K1; 5'-GTCCTGCCCGTCACCGAGAT-3'. 40 cycles at 50 °C for 30 s, at 72 °C for 4 min, and at 94 °C for 30 s.
A second round of PCR was done with 2 µl of the first PCR reaction at an annealing temperature of 55 °C using nested primers; RB-K2; 5'-ATTTCACGGGTTGGGGTTTCTACA-3' LB-K2; 5'-GCCAGGTGCCCACGGAATAGTTTT-3'.
A 2.6 kb band was isolated and sequenced with the LB-K2 and RB-K2 primers. This band was cloned in pUC18 using the Sureclone Ligation Kit (Pharmacia).
Complementation of the mutant
A 3.1 kb fragment containing approximately 2.1 kb of the EPC1 genomic sequence, and a 1kb upstream sequence was amplified from 100 ng WT genomic DNA using primers; H1-L: 5'-CGCAACAAAAACGATGAAGA-3' H1-R: 5'-CCGTCCATAGCTAGAACGTGA-3'. 40 cycles at 60 °C for 30 s, at 72 °C for 3 min, and at 94 °C for 30 s.
The fragment was isolated, sequenced and cloned into the binary vector pGPTV-HPT (Becker et al., 1992). This was transformed into Agrobacterium tumefaciens GV3101. Epc1-2 mutant plants were transformed by floral dip and transformed seeds were selected on MS agar plates containing 2% sucrose and 20 µg ml–1 hygromycin.
Golgi localization
The EPC1 genomic coding region was amplified using primers; LPTYFP5': 5'-TGGCGCGCCAATGGGAGGAGGAGAAGTTA-3' LPTYFP3': 5'-TGGCGCGCCCCAGAACCATAAATTGCG-3'.
The fragment was cloned as a 5' translational fusion to the eYFP reporter gene with expression being driven by the 35S promoter. This cassette was cloned between the left and right T-DNA borders of pGREEN II (Hellens et al., 2000) which carried the NPTII gene for selection of transformed plants. This plasmid was transformed into the Agrobacterium strain LBA4404 which had previously been transformed with the pSOUP helper plasmid. Agrobacterium transformed with these reporter constructs were grown to an O.D. of 1.0 and infiltrated into tobacco leaves using a syringe. After 3 d, imaging of YFP fluorescence was performed by using a Zeiss LSM 510 confocal laser scanning microscope.
RNA extraction and northern blot analysis
Leaf tissue was frozen in liquid nitrogen and used directly for RNA extractions. Approximately 100 mg frozen leaf tissue from WT and mutant plants was homogenized in 1 ml TRIzol Reagent (Invitrogen, Paisley, UK). Following the addition of 0.2 ml chloroform, each sample was centrifuged (6000 rpm) at 4 °C for 10 min and the supernatant removed. RNA was precipitated with isopropyl alcohol and quantified. 30 µg RNA was used for northern analysis.
EPC1 and 18S gene probes were amplified from WT genomic DNA by PCR using the following primers; EPC1; 5'-GAAAGGTTATACACTTCTGAT-3' and 5'-CGATTTTTCAGGCCAATGCACA-3' 18S; 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3'. Northern blots were visualized using a PhosphoimagerTM:SI (Molecular Dynamics, Sevenoaks, UK) and quantified using the ImageQuant® program (Molecular Dynamics).
PACE analysis of polysaccharides
Plant materials were ground in a ball Mixer Mill MM200 (Glen Creston, Middlesex, UK) and the cell walls prepared according to Goubet et al. (2002). Aqueous suspensions were prepared by homogenization in a glass potter before analysis.
The analysis of HGA, galactan, and arabinan from cell walls was performed as described in Barton et al. (2006) with modifications for arabinan and galactan. Cell wall material (0.5 mg) was incubated with enzyme in a total volume of 0.5 ml for 4 h. After the reaction, the solutions were boiled and dried. The derivatization was modified by using less ANTS (0.02 mM) and NaCNBH3 (0.2 mM) in a total volume of 40 µl of DMSO/water/acetic acid (20/17/3 by vol.). After the derivatization, the samples were resuspended in 30 µl of 2.4 M urea and 2 µl was loaded onto a polyacrylamide gel. Endo-PG-released oligosaccharides were derivatized with AMAC as described in Barton et al. (2006). Each biological sample was studied using multiple cell wall preparations, digests, and gels, to yield quantification from at least ten gel lanes in total. Standards were run alongside samples in each gel to obtain a standard curve for quantifying sugars in the samples. For AMAC-derivatized oligosaccharides GalU and (GalU)3 were used as a standard curve. For ANTS-derivatized oligosaccharide fingerprints, galactose, arabinose, and ß-(-1,4)-mannotriose standards were used.
Extraction and analysis of RG-II
Extraction of pectic material and further purification of RG-II was performed as described by Ishii et al. (2001). RG-I and RG-II polysaccharides were purified from endo-PG-soluble material by size-exclusion chromatography on a Superdex-75 HR 10/30 (Amersham Pharmacia Biotech Inc., Uppsala) column eluted at 0.6 ml min–1 with 50 mM ammonium formate, pH 5.3. RG-I, RG-II (monomer and dimer), and oligogalcturonide fractions were manually collected. Each fraction was dialysed and freeze-dried as above.
Fixation and analysis of hypocotyls
Hypocotyl tissue was harvested from plants grown for 12 d on MS agar plates. The plates were orientated at 90° so that the hypocotyls were in constant contact with the agar [growth conditions used by Singh et al. (2005) for their analyses]. Tissue was fixed for 3 h at room temperature (RT) in 4% formaldehyde, 4% glutaraldehyde, and 50 mM sodium phosphate buffer and dehydrated through an ethanol series. Hypocotyls were embedded in LR white resin at RT, and allowed to polymerize at 60 °C (Handford et al., 2003). Sections of 0.2 µm were transferred to Formvar-coated glass microscope slides and stained with 1% toluidine blue. Stained tissue was viewed using a light microscope (Olympus IX70) and pictures were taken using a Camedia C-3030 digital camera (Olympus). Calibration was obtained under identical magnification and used to analyse the diameter of the hypocotyls and, with the aid of the JavaImage program, the size of the cells within each of the three cortical cell layers. Sections of the hypocotyls showing 14–16 cells in the outermost cortical cell layer were routinely used to ensure that comparable regions of the hypocotyls were measured.
Immunolocalization
The sections were labelled according to Handford et al. (2003), except that 2% fetal calf serum in phosphate-buffered saline (PBS) was used for 5 min at room temperature before incubation for 1 h with the secondary antibody. Antibodies (rat-JIM13 and mouse-CCRC-M7) were diluted 1:20 in the blocking buffer. The secondary antibodies (cy3 anti-rat or anti-mouse) were diluted 1:200 in blocking buffer. The slides were washed with PBS, stained in Calcofluor white (0.1 mg ml–1 in PBS buffer) for 5 min and then washed with water. Slides were observed using an Olympus BX61 microscope.
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Results |
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Isolation of the epc1-2 mutant
A dwarf mutant was isolated from a screen for stature mutants from the INRA-Versailles Arabidopsis T-DNA tagged population (ecotype Ws). Southern analysis using a probe to the T-DNA showed a single band in a number of digests (SacI, SmaI, and XhoI) (data not shown) indicating the presence of a single T-DNA insertion. Inverse-PCR was used to clone the sequences flanking both left and right borders of the T-DNA. Analysis of these sequences revealed that the T-DNA was inserted into the 4th exon of the EPC1 gene (Fig. 1). The EPC1 gene (At3g55830) was identified by Singh et al. (2005) who described their analysis of the epc1-1 mutant which is a Syngenta SAIL line (68a11) carrying a T-DNA insertion in the first intron of the Arabidopsis EPC1 gene in ecotype Columbia (Col). Like the SAIL line 68a11, the epc1-2 mutant is severely reduced in stature (Fig. 2). Leaf area, and stem and root elongation are all greatly reduced, and it has a slightly delayed flowering phenotype in both long and short photoperiods. In addition the epc1-2 mutant also displays early senescence (cotyledons start to senesce in the mutant after two true leaves have been formed), but it successfully completes a full life-cycle and produces viable seed.
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The epc1-2 mutation was shown to be recessive as the F1 backcross population all exhibited a non-dwarf phenotype whilst the F2 population demonstrated a normal phenotypic segregation of 213 non-dwarf to 72 dwarf, a predicted ratio of approximately 3:1. All dwarf plants were found to be phosphinotricin (PPT) resistant which supports linkage of the dwarf phenotype to the T-DNA carrying the BAR gene. A wild-type genomic fragment containing the complete EPC1 coding region (2.1 kb) plus 1 kb of upstream sequence was transformed into the epc1-2 mutant. Complete restoration of the WT phenotype in several transgenic lines confirmed that the mutant phenotype is caused by the T-DNA insertion into the EPC1 gene, and not by any underlying second-site mutations. Two of these complemented lines, epc1-2-C1 (shown in Fig. 2) and epc1-2-C2 were used in further studies.
Comparable levels of expression were detected by northern analysis of leaves (in both light and dark), roots, stem, and flowers of WT plants, but no significant expression was detected in these tissues of the epc1-2 mutant. In addition, there was no evidence from the northern, or from RT-PCR analysis, for the existence of a truncated message (up to the point of the T-DNA insertion) in the epc1-2 mutant indicating that this is likely to be a null mutant (Fig. 3; data not shown). WT levels of expression were detected in leaf tissue of each of the complemented lines.
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In addition to EPC1, two other genes encoding GT64 proteins are present in Arabidopsis (At1g80290 and At5g04500). The function of these genes remains unclear at present although it is clear from the dwarfed phenotype of the epc1 mutants that neither of the other two genes can functionally compensate for the inactivation of EPC1.
The epc1-2 mutant has reduced cell size and defective root hair development
Cell size is generally smaller in the epc1-2 mutant compared with wild-type plants. Epidermal cell lengths in the mutant were less than half the length of those in WT or epc1-2-C1 plants (Fig. 4A). Comparisons of WT and epc1-2 10-d-old seedlings highlighted a significant difference in the length of the primary root (even though the size of the cotyledons at this stage of development remained comparable). On average, the length of the primary roots of epc1-2 seedlings (0.6 cm) was about one-third of the length of seedlings of WT and complemented lines (1.7–1.8 cm). In addition to having a short primary root, the epc1-2 mutant demonstrates an obvious phenotypic abnormality in the number, length, and distribution of the root hairs. Consistent with the reduced shoot epidermal cell size, root hairs of the epc1-2 mutant are significantly shorter than WT and complemented mutant plants (Fig. 4B). Defects in cell elongation and enlargement are known to cause similar root hair phenotypes in the root hair defective 2 (rhd-2) and rhd-3 mutants (Schiefelbein and Somerville, 1990) both of which result in the production of short, stubby hairs similar to those observed for the epc1-2 mutant.
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Cell adhesion appears normal in the epc1 mutants
Mutations in enzymes that synthesize certain cell wall pectins result in reduced cell–cell adhesion as is observed in the tobacco nolac-H14 and the Arabidopsis qua1 mutants (Iwai et al., 2001; Bouton et al., 2002). Reduced cell adhesion was reported by Singh et al. (2005) for 12-d-old epc1-1 seedlings, although cell adhesion was normal in younger (6-d-old) epc1-1 seedlings. Detailed, comparative analysis of hypocotyls from 12-d-old seedlings of both the epc1-1 and epc1-2 mutants was performed. Sections of the hypocotyls from both mutants and their respective parental ecotypes, Ws and Col, were studied using light microscopy. The number of cells within each of the three cortical cell layers was determined and their size calculated using the JavaImage program. The diameter of the hypocotyls was measured using a standard calibration slide. Sections varied in size and cell number depending upon the position along the length of the hypocotyls. Therefore, to ensure that comparable sections were examined, only sections having between 14–16 cells in the outermost cortical cell layer were analysed. The analysis was repeated for a number of seedlings from each genotype so that four sections (approximately 20 µm apart) each from three blocks of embedded tissue were examined. The integrity of cellular adhesion varied between the 12 sections of hypocotyls from the same genotype, even for Ws and Col seedlings, which may be due to the embedding and sectioning process. Sections showing the greatest and least cell adhesion from each genotype are shown in Fig. 5A–H. It was not evident from this analysis that the epc1-1 or epc1-2 mutants had significantly reduced cell adhesion compared with WT control tissues.
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The diameter of the hypocotyl of the epc1-1 mutant was also reported to be much larger than WT at 12 d after germination (Singh et al., 2005). Average hypocotyl diameters of 12 seedlings of both mutants and their respective WT ecotypes indicated a slightly larger average diameter for both the epc1-1 and the epc1-2 mutants, although the difference was not significant (Fig. 5I). It remains unclear, at present, as to why the observations of Singh et al. (2005), of reduced cellular adhesion and 5-fold increase in hypocotyl diameter for the epc1 mutants cannot be repeated. However, as no significant differences in cell adhesion or hypocotyl diameter were observed, it would appear that they are not linked to the epc1 mutations or the dwarf phenotype of the mutants. Our observations suggest that EPC1 is therefore unlikely to be involved in cell–cell adhesion but in some other process that is critical for normal plant growth and development.
The epc1-2 mutant exhibits an altered response to ABA
Since reduced stature, such as that observed in the epc1-2 mutant, is often caused by reduced biosynthesis of, or response to, gibberellic acid (GA), the response of the epc1-2 mutant to exogenous GA was tested. Twice weekly application of 10 µM GA could not rescue the dwarf phenotype of epc1-2 indicating that GA biosynthesis was not affected in the mutant. In vitro assays were then used to investigate the response of the mutant to GA. Endogenous GA biosynthesis was inhibited by paclobutrazol (1 µM) and exogenous GA was supplied at concentrations between 0 µM and 100 µM. Hypocotyl elongation of the mutant was comparable with WT plants, suggesting that the epc1-2 mutant does not have an altered response to exogenous GA (Fig. 6A). However, a 10-fold higher level of exogenous GA was required by the epc1-2 mutant to achieve comparable levels of germination to WT plants (Fig. 6B). This demonstrates that whilst epc1-2 responds normally to GA, higher levels of GA than normal are required for germination. As GA is known to act antagonistically to ABA in seed germination, the sensitivity of the epc1-2 mutant to ABA was investigated by measuring germination on media containing norflurazon (a carotenoid biosynthesis inhibitor used to inhibit endogenous biosynthesis of ABA in the seed) with or without exogenous ABA. Figure 6C shows that germination, as measured by radicle emergence, of the epc1-2 mutant is more sensitive to ABA than WT. This increased sensitivity is also seen in the effect of ABA on root elongation, where ABA inhibits the elongation of epc1-2 roots to a greater extent than that of WT roots (Fig. 6D). The increased sensitivity to ABA of the epc1-2 mutant could explain why higher levels of GA are required to stimulate seed germination to the same levels as WT seed.
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As the mutant exhibited an altered response to ABA, but not to GA, it was examined whether it had altered sensitivity to other hormones or whether it was specifically affected in its response to ABA. The response to auxin was tested by measuring the inhibition of root growth by increasing concentrations of both indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA) (0–0.5 µM). In each case no significant differences between epc1-2 mutant and WT plants were detected. Similarly, a comparable response to ethylene was observed in epc1-2 and WT plants, epc1-2 demonstrated a normal triple response when grown in the dark, and there was no difference in the inhibition of root growth on 1-aminocyclopropane-1-carboxylic acid (ACC) (0–100 µM). Furthermore, the epc1-2 mutant shows a normal hypocotyl elongation response to epi-brassinolide, application of which failed to rescue the mutant phenotype. Thus, the epc1-2 mutant is not affected in its response to all hormones, but shows a specific hypersensitivity to ABA. The application of exogenous ABA or auxin had no significant effect on the level of EPC1 gene expression (Fig. 3).
EPC1 is likely to be localized to the Golgi apparatus
To investigate the subcellular localization of the protein, YFP was fused to the C-terminus of EPC1 under the control of the CaMV 35S promoter. This construct was transformed into Agrobacterium and infiltrated into tobacco leaves. After 3 d, the localization of the fusion protein was examined by laser scanning confocal microscopy of the leaf epidermal cells. The fluorescence was observed in small punctuate structures within the cytoplasm (Fig. 7) which were highly motile (data not shown). The size and number of the structures were typical of what is observed for the Golgi apparatus. This supports recent proteomic observations that EPC1 co-fractionated in subcellular fractionation experiments with known Golgi apparatus proteins (Dunkley et al., 2006), and is consistent with the predicted structure of EPC1 as a type II membrane Golgi-localized glycosyltransferase. Most glycosylation products of the Golgi apparatus are integrated into the plasma membrane, or secreted into the extracellular matrix. Therefore, the site of action of the EPC1 glycosylation product is likely to be at, or outside, the plasma membrane.
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Some pectic components of the cell wall are altered in the epc1-2 mutant
Synthesis of cell wall components involves the action of many glycosyltransferases that are either located in the plasma membrane (cellulose synthase and callose synthase), or in the Golgi apparatus. As EPC1 is a putative retaining glycosyltransferase, and as the GFP localization is consistent with it being in the Golgi apparatus, it is possible that the mutation may affect the synthesis of a cell wall polysaccharide.
Analysis of some of the pectic components of the cell wall, namely HGA, -(1,5)-arabinan (present in both AGPs and rhamnogalacturonan I (RG-I)), and ß-(1,4)-galactan (present in RG-I), was carried out using PACE (polysaccharide analysis by carbohydrate gel electrophoresis) of leaf material from epc1-2, WT, and epc1-2-C1 plants. In this technique, oligosaccharides released from the cell wall by the action of a pure and specific hydrolase are derivatized by a fluorophore and the relative quantities of the different oligosaccharides are analysed by polyacrylamide gel electrophoresis (Goubet et al., 2002). As endo-polygalacturonase (endo-PG) cannot hydrolyse highly esterified HGA, an alkaline pre-treatment is necessary to give access to all the HGA (Goubet et al., 2003). The PACE revealed that the quantity of unesterified HGA in epc1-2 was unchanged relative to WT and, furthermore, that the same quantity of total HGA was found after alkaline treatment (Fig. 8). The quantity of ß-(1,4)-galactan in epc1-2, however, was only about half of that present in WT cell walls (Fig. 8). The reduced level of ß-(1,4)-galactan observed in the epc1-2 mutant fits with the reduced galactose levels observed in the epc1-1 mutant (Singh et al., 2005). The monoclonal antibody, LM5, which recognizes tetramers of ß-(1,4)-galactan was used in immunolocalization studies to investigate whether the distribution of galactan was altered in resin-embedded sections of the epc1-2 mutant, but no differences were observed (data not shown). In contrast to the reduced levels of galactan, the quantity of -(1,5)-arabinan in the epc1-2 mutant was comparable with WT plants. Complemented mutant plants exhibited WT levels of HGA, -(1,5)-arabinan and ß-(1,4)-galactan (data not shown).
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To investigate whether there were any differences in levels or composition of RG-II in the mutant, RG-II was extracted and purified as described by Ishii et al. (2001). RG-I is the main polysaccharide and RG-II is a minor polysaccharide in both WT and mutant cell walls. RG-II was mainly present as the borate cross-linked dimer form and the ratio between RG-II monomers and dimers was similar in both the WT and the mutant. The monosaccharide composition of the RG-II dimer was determined by GC of the trimethylsilyl methyl ester methyl glycoside derivatives (York et al., 1986) and was also found to be similar between mutant and WT (data not shown).
As AGPs have been implicated in the response of roots to ABA (van Hengel and Roberts, 2003), and as inhibiting AGP function using ß-glucosyl Yariv reagent inhibits cell division and elongation resulting in a dwarf phenotype similar to the epc1 mutants (Willats and Knox, 1996), AGP levels and distribution were examined by immunolocalization. Immunofluorescence of stem, hypocotyls, and root and leaf sections using two monoclonal antibodies JIM13 (Yates et al., 1996) and CCRC-M7 (Steffan et al., 1995) which recognize some AGP epitopes showed that these epitopes were present and their distribution was not altered in the epc1-2 mutant (data not shown). These results suggest that there is no substantial change in AGP distribution or abundance in the epc1-2 mutant, although it is difficult to rule out the possibility that there is a change in levels or structure of a particular AGP.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this paper, the characterization of a second mutant allele of the EPC1 gene, which encodes a putative retaining glycosyltransferase of the GT64 family, is described. Our observations do not support a role for EPC1 in cell adhesion as proposed by Singh et al. (2005). Interestingly the epc1-2 mutant exhibits hypersensitivity to ABA, but not to any of the other hormones tested, raising the possibility that it may be involved in plant growth responses to ABA.
The epc1-2 mutation is in a putative Golgi-localized glycosyltransferase
Complete complementation of the epc1-2 mutation by the EPC1 gene and upstream sequence showed that the T-DNA insertion in this gene is responsible for the phenotype of this mutant. Localization of the EPC1–YFP fusion protein to punctate structures typical of the Golgi apparatus supports the prediction that EPC1 is present in this organelle. This view is also supported by recent proteomic observations that EPC1 co-fractionated in subcellular fractionation experiments with known Golgi apparatus proteins (Dunkley et al., 2006).
EPC1 exhibits homology to the mammalian EXT and Drosophila TTV proteins. The EXT and TTV proteins produce heparan sulphate chains which are attached to proteoglycans and become localized at the cell surface. These heparan sulphate proteoglycans (HSPGs) are known to act as co-receptors or internalization receptors for signal molecules such as growth factors (Lander and Selleck, 2000). Heparan-related polysaccharides have not been found in plants. However, the cell wall contains pectic polysaccharides with a similar structure of a disaccharide motif with alternating charged and uncharged monosaccharides (GalU-Rham in RG-I), and signalling functions in both plant defence and plant development have been identified for some of these oligosaccharides (Ridley et al., 2001; Navazio et al., 2002). Plant AGPs are a diverse range of proteoglycans located in Golgi-derived vesicles, the plasma membrane, extracellular matrix, and the cell wall, and could be thought of as plant equivalents of mammalian HSPGs. Glycosylation of AGPs is thought to occur in the Golgi and involves the attachment of large, highly branched galactan chains decorated with arabinose. A lot of biochemical, immunohistochemical, and molecular evidence indicates that AGPs are involved in different aspects of plant growth and development, including cellular differentiation, xylem development, cell division, programmed cell death, and root epidermal cell growth, as well as GA and ABA signalling (Suzuki et al., 2002; van Hengel and Roberts, 2003; van Hengel et al., 2004). Whilst differences were not observed in AGP levels or distribution in immunolocalization experiments using JIM13 and CCRC-M7 antibodies, AGP structures are so diverse that it is possible that the structure of a particular AGP may be altered in the mutant.
Effect of the epc1-2 mutation on the response to ABA
As the epc1-2 mutation results in an increased sensitivity to ABA and not to any of the other hormones tested, EPC1 must negatively affect the response to ABA. There is now plenty of evidence for one or more pathways that negatively regulate ABA responses. At least one of these pathways involves a plasma membrane-associated step as the ROP10 GTPase needs to be associated with the plasma membrane to be functionally active (Zheng et al., 2002). EPC1 may be involved in the glycosylation of a protein or polysaccharide located on or outside the plasma membrane which has an effect on ABA signalling. One possibility is that the glycosylation mediated by EPC1 results in either the inactivation of a positively acting factor, or the activation of a negatively acting factor in the ABA response pathway. In the epc1-2 mutant, these factors would not be glycosylated, resulting in an exaggerated ABA response.
The hypersensitivity to ABA could explain the dwarfed phenotype of the epc1-2 mutant. Such a phenotype has been observed in another ABA hypersensitive mutant, ahg2-1 (ABA hypersensitive germination), which exhibits a significantly shorter hypocotyl, stem, and main root (Nishimura et al., 2005). ABA is known to inhibit growth both by limiting cell extensibility (Kutschera and Schopfer, 1986), and by arresting cell division at the G1 phase of cell cycle (Liu et al., 1994). This latter effect of ABA may be mediated through an ABA-inducible cyclin-dependent protein kinase inhibitor (ICK1) that interacts with Cdc2a and CycD3 (Wang et al., 1998). Increased levels of ICK1 cause an inhibition of cell division resulting in dwarf plants (Wang et al., 2000). The increased ABA sensitivity could thus cause the reduced stature of the epc1-2 mutant by either, or both, of the above mechanisms. ABA is also known to be required during root regeneration where the perception of ABA is mediated by an AGP, AtAGP30 (van Hengel and Roberts, 2003; van Hengel et al., 2004).
Effect of the epc1-2 mutation on cell wall pectin
EPC1 encodes a putative retaining glycosyltransferase. The backbones of the pectic polysaccharides RG-I, RG-II, and HGA are produced by retaining enzymes, as are some of the linkages in the side chains of RG-II. HGA quantity and esterification, and levels and composition of RG-II were studied, but changes in either HGA or RG-II were not detected. The only difference in pectic cell wall components detected between epc1-2 and WT plants was in ß-(1,4)-galactan, a component of RG-I. As EPC1 is a putative retaining glycosyltransferase, it is unlikely to be directly involved in the synthesis of ß-(1,4)-galactan which would require an inverting enzyme. However, it may be possible that EPC1 is involved in the synthesis of part of the RG-I backbone which is required for the galactan side chain.
How the reduced levels of ß-(1,4)-galactan caused by the epc1-2 mutation relate to the dwarfed stature and altered response to ABA is not yet clear. The presence of RG-I associated ß-(1,4)-galactan and -(1,5)-arabinan epitopes has been correlated with specific developmental processes (Willats et al., 1999; Bush et al., 2001), although the functions of the different RG-I side-chains are unknown. In the Arabidopsis root apex ß-(1,4)-galactan was shown to occur in the transition zone where cells are undergoing their final divisions and starting to elongate more rapidly (McCartney et al., 2003). Reduced levels of ß-(1,4)-galactan in the transition zone have been correlated with reduced cell division and elongation, thus resulting in reduced cell size. It has been shown that ß-(1,4)-galactan levels in the root transition zone are reduced by mutations, or by auxin and cytokinin treatments, that inhibit cell elongation and root growth (McCartney et al., 2003). It may be that the reduced levels of ß-(1,4)-galactan are a result of the reduced cell elongation caused by the increased response to ABA in the epc1-2 mutant. Alternatively, reducing the levels of ß-(1,4)-galactan may be part of the mechanism by which ABA limits cell extensibility, the increased response to ABA in the epc1-2 mutant would therefore exaggerate these effects.
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Acknowledgements |
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We are grateful to the Station de Génétique et d'Amélioration des Plantes INRA, Versailles, France and to NASC (Nottingham, UK) for the production and distribution of the T-DNA mutant population. We thank Harry Gilbert (University of Newcastle, UK) for providing us with
-(1,5)-arabinanase and ß-(1,4)-galactanase. We thank Syngene (Cambridge, UK) for the gift of GeneTools software. We are grateful to T Weimar for his help in obtaining the confocal YFP images. This work was funded by grants from the BBSRC (UK). AGD was funded by Novozymes (Denmark). TI is the recipient of a grant from BRAIN (Japan). |
Footnotes |
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* Present address: National Agricultural Research Center for Western Region 2575 Ikano, Zentsuji, Kagawa 765-0053, Japan.
Present address: Bayer BioScience NV, Technologiepark 38, B-9052 Gent, Belgium.
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