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First published online August 25, 2006; 10.1104/pp.106.084244 Plant Physiology 142:458-470 (2006) © 2006 American Society of Plant Biologists Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis1,[W]Department of Plant Biology, Carnegie Institution, Stanford, California 94305 (J.M.E., N.K., C.S.); Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701 (M.J.K.); and Department of Biological Sciences, Stanford University, Stanford, California 94305 (C.S.)
A series of gene constructs encoding synthetic glycomodule peptides with N-terminal signal sequences and C-terminal green fluorescent proteins were expressed in transgenic Arabidopsis (Arabidopsis thaliana) under the control of the 35S promoter. The synthetic glycomodule peptides were composed of repetitive proline-containing motifs that have been previously found to be substrates for prolyl hydroxylases and subsequent O-glycosylation of the hydroxyproline residues. All of the constructs were secreted in aerial tissues, but not in roots. The amount of hydroxylation and glycosylation of the various constructs varied depending on the tissue. Also, accumulation of the proteins exhibited a high degree of cell-type specificity within various tissues due to posttranscriptional effects. The observations reveal a high level of complexity in the synthesis, secretion, and turnover of the glycoproteins.
Arabinogalactan proteins (AGPs), Pro-rich proteins, and extensins belong to a large group of glycoproteins, known as Hyp-rich glycoproteins (HRGPs), that are analogous to animal proteoglycans. These complex molecules are the major Hyp O-glycoproteins expressed throughout the plant kingdom from unicellular green algae (Roberts, 1974  ; Ertl et al., 1992; Kieliszewski and Lamport, 1994) and bryophytes (Lee et al., 2005) to angiosperms (Schultz et al., 1998; Gaspar et al., 2001; Showalter, 2001). All HRGPs are located outside, or attached to, the plasma membrane. The proteins are defined by posttranslational modifications that include the hydroxylation of some Pro residues by prolyl 4-hydroxylases (P4Hs) in the endoplasmic reticulum (ER) and glycosylation of Hyp with short arabinooligosaccharides or larger AG polysaccharides (Lamport, 1965a, 1965b; Pope, 1977). The Hyp contiguity hypothesis predicts that glycan addition to Hyp is directed by the protein sequence: Regions of contiguous Hyp residues are typically arabinosylated, whereas larger AG glycans occur mainly on clustered, noncontiguous Hyp residues (Kieliszewski et al., 1992; Shpak et al., 1999, 2001; Kieliszewski, 2001).
The Arabidopsis (Arabidopsis thaliana) genome encodes 11 putative P4Hs (Yuasa et al., 2005
HRGPs have been proposed to be involved in many aspects of growth and development, ranging from wall architecture and assembly to cell proliferation, cell-to-cell recognition, and cell expansion (Lamport, 1965
One of the many challenges in understanding the synthesis and function of HRGPs is that they are typically very highly glycosylated. The high degree of glycosylation combined with the large number of species makes it difficult to purify a specific HRGP for structural analyses. Similarly, the large number of proteins carrying similar or identical glycans makes it challenging to use mutant analysis to infer biological function. The use of synthetic genes combined with an enhanced green fluorescence protein (EGFP) tag to express single repetitive glycopeptide and peptide motifs of AGPs and extensins allows detailed characterization of HRGP glycan structures by simplifying their isolation and characterization (Shpak et al., 1999
Expression of SynGMs in Arabidopsis
Ten synthetic gene constructs based on two different types of Hyp-rich glycomodules (Shpak et al., 2001
Roots (from 5-d-old seedlings), stems, leaves, flowers, and siliques of transgenic plants carrying the various constructs were screened by western blotting using monoclonal anti-EGFP as a primary antibody (Fig. 2 ). Several independent lines were characterized for each construct to ensure that the results were characteristic of the glycomodule rather than being caused by anomalous insertion events. SDS-PAGE of extracts from transgenic lines for the (SPPP)15, (SPPPP)18, (YK)20, (VP)11, (SP)32, (GAGP) 3, (TP)101, and LeAGP1 constructs accumulated fusion proteins with higher apparent molecular mass than that predicted for the nonmodified polypeptides encoded by the respective SynGMs. This was due to extensive O-glycosylation of the fusion proteins. However, none of the 60 to 80 transformed plants with the repetitive (AP)51 motif (Tan et al., 2003 ) or the (SPP)24 motif (Shpak et al., 2001) exhibited any detectable expression on western blots, although the constructs had previously been observed to result in glycoprotein production in tobacco cell cultures.
For each SynGM, the relative molecular mass of the glycoproteins on western blots varied from one organ to another (Fig. 2). The incremental shifts in relative molecular mass from the expected size of the peptide before and after posttranslational modifications (i.e. O-glycan addition) are shown in Table I . This is also supported by Pro hydroxylation and the sugar composition of the purified SynGMs (see later; Table II). For example, for the (SPPP)15 contsruct, two bands were detected in several tissues at 60 to 70 kD and 90 to 100 kD, but only one band of approximately 80 to 100 kD was observed in leaves. Because the expected unmodified (nonhydroxylated and nonglycosylated) relative molecular mass of the (SPPP)15-EGFP fusion polypeptide was 32.7 kD, approximately 36 and 65 kD of glycans were apparently attached to Hyp residues. Similarly, most of the other SynGMs were highly glycosylated.
Comparisons on the relative molecular mass of the SynGMs in Arabidopsis with similar experiments conducted in tobacco indicate that, in most cases, the relative molecular mass of the SynGMs expressed in Arabidopsis differ significantly from those observed in tobacco (Table I). For instance, the (VP)11 construct had a relative molecular mass of 45 to 52 kD in roots and leaves and approximately 65 kD in flowers. By contrast, Tan et al. (2003) reported a relative molecular mass of about 33.7 kD in tobacco Bright-Yellow 2 (BY2) cells due to addition of arabinosides and polysaccharides (Table I).
LeAGP1 was the only SynGM studied where the glycosylation pattern was similar among the plant organs and similar to expression in BY2 cells, showing only one broad band in the range of 130 to 230 kD. When expressed in BY2 cells, the hydroxylated polypeptide was approximately 43.3 kD and the glycosylated fusion approximately 164 kD. Thus, the AG in Arabidopsis are much larger than in BY2 cells. LeAGP1 expressed in Arabidopsis plants seems to have less dispersion and higher molecular mass compared with the smear of 37 to 105 kD found in the vegetative organs of tomato (Gao and Showalter, 2000
For all the SynGMs analyzed by immunoblotting, a band at 27 kD was frequently detected and probably represents EGFP that is cleaved from the fusion protein upon heat treatment as previously reported (Xu et al., 1999 ; Sun et al., 2004). To test this idea, an (SPPP)15 extract (EGFP on the C terminus; see Fig. 1) was treated at different temperatures from room temperature (with and without 8 M urea) to 100°C before loading into a gel (Fig. 2B). The expected relative molecular mass of the non-hydroxylated/glycosylated SPPP protein without the signal peptide is 33.0 kD. However, when incubated at 60°C or higher, the EGFP is cleaved from the protein and, at 70°C and above, most of EGFP is detected at the 27-kD band. Similar results (data not shown) were obtained using the same procedure with LeAGP1, although the EGFP fusion is on the N terminus of the glycoprotein.
Six SynGMs [(SPPP)15, (SPPPP)18, (YK)20, (SP)32, (VP)11, and LeAGP1] were extracted from leaves and partially purified by anion-exchange chromatography (except YK20; Supplemental Fig. S1). The elution pattern of the SynGMs with AGP motifs [(SP)32 and (VP)11] almost completely overlapped with that of the endogenous AGPs and, possibly, with other soluble polysaccharides. By contrast, SynGMs with extensin motifs [(SPPP)15 and (SPPPP)18] eluted partially or completely after the endogenous AGPs (Supplemental Fig. S1). In a second step of the purification procedure, the SynGMs from the anion-exchange chromatography together with the SynGM (YK)20 were purified on an affinity column containing monoclonal anti-EGFP antibodies.
Based on measurements of amino acid composition, the extent of Pro hydroxylation was determined for the purified SynGMs (Table II). The AGP motifs (VP)11, (SP)32, LeAGP1, and the extensin sequence (SPPP)15 expressed in Arabidopsis contained similar amounts of Hyp (59.0%, 95.3%, 94.7%, and 71.0% of original Pro units, respectively) to that observed previously in BY2 cells (Shpak et al., 1999
The ratio of protein to carbohydrate content (w/w) determined for six purified SynGMs was used to estimate the relative molecular mass contribution of the O-glycans attached to each synthetic peptide and compared with those observed on western blots (Table I). Direct measurement of glycan mass was consistent with an increased relative molecular mass of the SynGMs due to glycan addition on SDS-PAGE (Fig. 2). For most of the SynGMs, the calculated relative molecular masses were similar compared with the apparent relative molecular mass detected on western blots, but lower in the cases of the extensin motifs (SPPP)15 and (SPPPP)18 (Table I; Fig. 2). This is probably due to an overestimation of mass on western blots due to the poor mobility of the highly glycosylated peptides in SDS gels due to the steric hindrance of SDS binding to the polypeptide backbone (Hames, 1990
EGFP expression of fusion protein in seedlings from 1 d after germination (dag) to 5 dag was followed using CLSM. Fusion proteins were highly expressed only in roots from 1 to 5 dag (Fig. 3) for all constructs studied, except for control plants where EGFP was detected in all tissues. In the SynGM plants, the signal was absent or very weak in hypocotyls, cotyledons, or in the first leaves in many of the lines. The lines expressing LeAGP1 were the only ones in which EGFP was readily detected in hypocotyls and leaves (Fig. 2A). Thus, the presence of the glycomodules affected the accumulation of the EGFP fusion proteins in seedlings. To test whether the apparent absence of fusion proteins in some tissues might be due to suppression of the fluorescence of EGFP by posttranslational modification (i.e. misfolding), a western analysis was conducted on extracts of the aerial parts of the seedlings and compared with those obtained in roots (Fig. 4A). However, no band corresponding to EGFP or the SynGM proteins was detected in most of the samples from hypocotyls and leaves analyzed, with the exception of the LeAGP1 plants and the positive controls. This result is in agreement with CLSM observations (Fig. 3). To determine whether expression of SynGMs was regulated at transcriptional or posttranscriptional levels in the aerial parts of the seedlings, mRNA corresponding to a fragment of EGFP was visualized using reverse transcription (RT)-PCR analysis (Fig. 4B). The results indicated that all of the constructs were transcribed at high levels comparable to the actin control. Thus, the discrepancy between the steady-state levels of mRNA and the cognate protein indicates some posttranscriptional control of SynGM protein accumulation.
Expression of the SynGMs was examined by CLSM in cross sections of leaves and stems of 6-week-old plants to determine whether there was tissue-specific expression of the SynGMs. In leaves, expression was restricted to certain tissues (Fig. 5 ). Moreover, expression was patchy and not uniform, especially in the upper or lower epidermis and in stomata during early stages of leaf development (data not shown). In all plants studied, proteins were localized in both upper and lower epidermal cell layers. In (SP)32, (SPPP)15, and (GAGP)3 SynGMs, proteins were localized in vascular tissues, specifically in xylem and procambium cells (Fig. 5). Also, in (YK)20, the fusion proteins were localized at the boundaries of mesophyll cells.
SynGM accumulation in stems also exhibited tissue-specific patterns that varied from one construct to another (Fig. 5). In most cases, the proteins were localized in epidermal cells [(SPPPP)18, (YK)20, GAGP, and LeAGP1], but also accumulated in procambium/cambium cells [(SP)32 and (YK)20], primary xylem [(SP)32 and (SPPP)15], and secondary xylem, mainly in metaxylem elements [(SP)32 and LeAGP1] and cortical cells [(VP)11 and (SPPP)15]. The tissue expression of LeAGP1 in Arabidopsis stems is similar to that described for tomato stems (Gao et al., 1999 ; Gao and Showalter, 2000), although the epidermal cells were not reactive in the latter with the polyclonal PAP antibody (reacts against the Lys-rich subdomain of the Le-AGP1 protein backbone).
To determine the subcellular localization of the fusion proteins in epidermal cells in leaves (Fig. 6A ) and root seedlings (Fig. 6B), they were plasmolyzed with 500 to 800 mM mannitol. In this way, secreted proteins can be observed in the space that is formed between the plasma membrane and the cell wall.
In the epidermal cells of the leaves, all of the SynGMs were secreted to the cell wall, whereas EGFP with a tobacco signal sequence was not secreted. Although all the SynGMs were mostly secreted, some of the signal remained in the cytoplasm, the ER, and the nucleus (Fig. 6, A and B). The secretion of the SynGMs, but not the control protein, in the epidermal cells indicates that either glycosylation is necessary for secretion of proteins fused to the tobacco extensin signal sequence or the signal sequence needs to be separated from EGFP by an intervening peptide sequence to function.
Subcellular localization of SynGMs was variable along the roots (Fig. 6B). In most cases, such as (VP)11, (GAGP)3, and (YK)20 SynGMs, EGFP was intracellular and largely or entirely in the ER and nucleus. In plants expressing LeAGP1, which is glycosylphosphatidylinositol anchored, the EGFP was observed outside the plasma membrane, but also in the cytoplasm, indicating that it was partially secreted in both parts of the root analyzed (i.e. the differentiation zone and root tip). In two of the extensin motif proteins [(SPPP)15 and (SPPPP)18], the SynGMs were localized outside the membrane in close association with the cell wall, but also in the ER endomembrane system in the differentiation zone, whereas in the root tip they were detected only inside the plasma membrane. In the AGP-related protein (SP)32, EGFP was secreted in the root tip, but not in the differentiation zone (Fig. 6A). It seems that, in root cells, most of these SynGMs were retained in the ER secretory pathway. Interestingly, only the signal sequence-EGFP control was found mostly soluble in the cytosol (Fig. 6A). Also, the same construct was not secreted in tobacco cells (Zhao et al., 2002 Using CLSM to image the EGFP fusion proteins in the differentiation zone of the root where some types [(VP)11 and (SP)32, AGP motifs] were not secreted outside the plasma membrane, it was found that a large number of fusiform bodies were visible in the ER (Fig. 7, B and C ; VP data not shown). In the same type of cells where secretion of other glycoproteins [(SPPP)15 and (SPPPP)18 motifs and LeAGP1] takes place, much less or none of these bodies were found (Fig. 7, D and E). It seems that secretion of the SynGMs and the presence of the fusiform bodies in the ER are inversely related in epidermal root cells and the appearance of fusiform bodies appears to be a consequence of the overexpression of glycoproteins in the elongated root cells. In leaf epidermal cells, even when there was overexpression of glycoproteins, no fusiform bodies were detected in the ER in all lines studied (Fig. 7H). Thus, the conditions necessary for induction of fusiform bodies is different in root cells compared with epidermal cells in leaves.
Because of the large number of HRGPs in Arabidopsis, it is technically challenging to exploit the genetic tools available in Arabidopsis to study the synthesis and function of these intriguing glycoproteins. Our initial goal in producing a series of transgenic plants that express SynGMs was to develop a set of lines that would facilitate the analysis of single molecular species of HRGPs in Arabidopsis and also allow a comparison with tobacco. The characterization of the transgenic lines presented here highlights the considerable complexity of posttranslational modifications that these glycoproteins are subject to and suggests the existence of cell-type-specific effects on HRGP accumulation.
Analysis of the apparent molecular mass of a single SynGM expressed in many types of tissues showed that, in Arabidopsis, the same protein backbone can be posttranslationally modified in more than one way. Previous work in tobacco showed that the SynGMs used here become extensively and very specifically glycosylated in BY2 cells (Shpak et al., 1999 The cell-type-specific pattern of posttranslational modifications presumably reflects both cell-type-specific expression and substrate specificity of P4Hs and glycosyltransferases. Furthermore, in some cases, two different glycosylation patterns were found in the same tissue. For example, in seedling roots, the extensin (SPPP)15- and (SPPPP)18-containing proteins accumulated in two distinct relative molecular mass forms (Fig. 2). It is likely that the lower relative molecular mass band corresponds to arabinosylated SynGM and the higher mass band to the added presence of AG polysaccharides. This is corroborated by the monosaccharide composition on these two SynGMs, where not only Ara was found, but also Gal. Explanations for the anomalous glycosylation profiles of the extensin-based motifs follow.
(1) Incomplete hydroxylation of the polyproline blocks with values between 51% to 71% of the units in the extensin glycomodules [(SPPP)15, (SPPPP)18, and (YK)20] gave rise to a mixture of contiguous and noncontiguous Hyp residues and corresponding arabinosylation and arabinogalactosylation at these sites. Interestingly, (YK)20 belongs to the P3 type of extensin repeat (SOOOOSOSOOOOYYYK) that is the most common motif in many species (e.g. Medicago, tomato, tobacco, beans, etc.), but is totally missing from Arabidopsis extensins. Thus, it is possible that this motif is a poor substrate for the hydroxylases and glycosyltransferases in Arabidopsis. The in vivo partial hydroxylation of the extensin motifs is consistent with the incomplete in vitro hydroxylation of extensin sequences by AtP4H-1 and AtP4H-2 (Hieta and Myllyharju, 2002 (2) The codes for Hyp-O-glycosylation do not extend to Arabidopsis plants. However, this seems unlikely as expression of repetitive (SPP)20 and (SPPPP)18 in Arabidopsis cell cultures yielded transgenic proteins in which all Pro residues were hydroxylated and subsequently arabinosylated, but not arabinogalactosylated (J. Xu and M. Kieliszewski, unpublished data).
The results obtained in BY2 cells were ambiguous about the SPPP motif and in maize (Zea mays) the TOTO motif was only arabinosylated (Kieliszewski et al., 1990
Although the SynGMs were expressed under transcriptional control of the constitutive 35S promoter, there was a strong effect of cell type on SynGM accumulation. For instance, the SynGMs were not detectable in leaves and hypocotyls of developing seedlings, but were abundant in roots at the seedling stage. Analysis of mRNA levels of the various constructs in seedling leaves, cotyledons, and hypocotyls indicated that the mRNA was abundantly present, as expected for genes under the control of the 35S promoter. The implication is that, in some cell types, the SynGMs are not translated or, more likely, are translated and degraded. In adult tissues, protein accumulation was tissue specific and generally most pronounced in epidermal, procambium, cambium, and xylem cells. Furthermore, the accumulation pattern varied from one construct to another. Thus, it is apparent that there is not only cell-type-specific expression of endogenous HRGP genes (Kieliszewski et al., 1992
All of the SynGMs used here (i.e. SPPP15, SPPPP18, and YK20) have previously been expressed in tobacco BY2 cells (Shpak et al., 1999 Expression of the various SynGMs had relatively minor effects on leaf and stem growth and development. By contrast, expression of SynGMs caused a strong inhibition of seedling root growth that was correlated with reduced cell expansion and reduced size of the meristematic zone (J.M. Estévez and C. Somerville, unpublished data). The spatial expression of the SynGM molecules and their in vivo Mr was highly variable from tissue to tissue in some cases [i.e. (SP)32, (YK)20], and almost constant for others [i.e. (SPPPP)18, (LeAGP1)], indicating that the secretory pathway (responsible for their final Mr) used for each glycopeptide in the different cell types is not equivalent in some cases.
The expression profile of putative At-P4Hs (Zimmermann et al., 2005 In conclusion, the results presented here provide a framework for utilization of synthetic glycomodules as tools to dissect the mechanisms of HRGP synthesis in Arabidopsis. The results illustrate the importance of cell-type-specific mechanisms in the synthesis of the HRGPs that extend beyond transcriptional control of individual HRGP genes. The results also provide a point of direct comparison between Arabidopsis and tobacco that indicates a high degree of interspecies variability in the way in which SynGMs are processed. The obvious implication is that, whatever the roles of HRGPs, those roles can accommodate extensive structural diversity during species divergence.
Plasmid Construction and Agrobacterium Transformation
The plasmid constructions were reported elsewhere (Shpak et al., 2001
Plant Material For growth on agar, seeds were disinfected with 95% ethanol, rinsed with sterile water, and air dried. Seeds were then mixed with 0.15% agar and planted on 1.5% agar plates with Murashige and Skoog medium under continuous light (140–220 mmol m–2 s–1) at 23°C. In some cases, seedlings were transferred to soil and grown in greenhouse chambers under 16-h light/8-h dark conditions at 23°C.
For analysis of fusion protein transcriptional levels in seedlings, total RNA was isolated from cotyledons and hypocotyls of 5-d-old seedlings using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA was obtained from 1 µg of RNA using SuperScript III (Invitrogen) according to the manufacturer's instructions. PCR-specific primers used were for EGFP (sense 5'-CTACCCCGACCACATGAAGCAGCAC-3'; antisense 5'-GTCGGCCATGATATAGACGTTGTGG-3') and for actin1 At2g37620 (sense 5'-AGAGATCACTGCTTTGGCTCC-3'; antisense 5'-ATCCGTCATACTCTGCCTTTG-3').
Proteins were extracted using the method described in Martinez-Garcia et al. (1999)
HRGP was purified according to Schultz et al. (2000)
Between 70 to 400 mg of HRGPs were solubilized in water (3–10 mL) and partially purified by anion-exchange chromatography in a DEAE-Sephacel column (30 x 1.4 cm i.d.) using a 200-mL linear gradient from 0 to 3.0 M NaCl in aqueous solution. The column eluents were analyzed for carbohydrates by the phenol-sulfuric method using Gal as standard (Dubois et al., 1956
For in situ detection of the SynGMs on leaves and stem tissues, hand cross-sections were made immediately before imaging and mounted on coverslips in distilled water. Whole seedlings were observed directly on the microscope without sectioning. Confocal imaging was performed using an MRC 1024 laser-scanning confocal head (Bio-Rad) mounted on a Diaphot 200 inverted microscope (Nikon), a Zeiss 510 laser-scanning confocal microscope, and a Leica TCS SP2 AOBS. The objectives used were a 60x Nikon PlanApo water immersion (WI) 1.2 numerical aperture (Technical Instruments), a 40x Nikon PlanApo WI 0.9 numerical aperture, and a HCX PL APO 63X/1.2 W Corr/0.17 Lbd. Bl. objective. The samples were excited with two lasers (Ar/Kr and He/Cd) at the following wavelengths: 568 nm for propidium iodine (PI) or chlorophyll fluorescence (PF), 488 nm for EGFP, and emission at 585 nm for PI/PF, and 522/520 nm for EGFP. Three-dimensional reconstructions of image stacks were carried out using Image J version 1.34 software. All images were processed with Adobe Photoshop 7.0 (Adobe Systems) and assembled with Illustrator (Adobe Systems) software.
Hand-cut stem cross sections (200 µm in thickness) were stained with Toluidine blue O (0.05% [w/v], in 0.1 M HCl at pH 1.0) for referencing the tissue. Calcofluor white (0.1% [w/v]; Sigma) in aqueous solution (Krishnamurthy, 1999
The following materials are available in the online version of this article. Supplemental Figure S1. Isolation of the synthetic peptides (top) (VP)11, (SP)32, (SPPP)15, (SPPPP) 18, and LeAGP1 from Arabidopsis leaves by DEAE-Sephacel anion-exchange chromatography.
We gratefully acknowledge the assistance of Pliny Hayes in producing the transgenic plants. Received May 26, 2006; accepted August 20, 2006; published August 25, 2006.
1 This work was supported by a grant from the U.S. Department of Energy (grant no. DOE–FG02–03ER20133).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Chris Somerville (crs{at}stanford.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.084244 * Corresponding author; e-mail crs{at}stanford.edu; fax 650–325–6857.
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ABSTRACT
RESULTS
-glucuronidase reporter gene. All constructs were under transcriptional control of the cauliflower mosaic virus 35S promoter. The plasmids were delivered into Agrobacterium tumefaciens (strain GV3101) by electroporation. Plants of the Columbia-0 ecotype were transformed as described by Clough and Bent (1998)