Characterization of Synthetic Hydroxyproline-Rich ... - Plant Physiology

Characterization of Synthetic Hydroxyproline-Rich Proteoglycans with Arabinogalactan Protein and Extensin Motifs in Arabidopsis1[W] Jose´ M. Este´vez, Marcia J. Kieliszewski, Natalie Khitrov, and Chris Somerville* 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).

1

This work was supported by a grant from the U.S. Department of Energy (grant no. DOE–FG02–03ER20133). * Corresponding author; e-mail [email protected]; fax 650–325– 6857. 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 ([email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.084244 458

The Arabidopsis (Arabidopsis thaliana) genome encodes 11 putative P4Hs (Yuasa et al., 2005), but only two, P4H-1 and P4H-2, have been characterized (Hieta and Myllyharju, 2002; Tiainen et al., 2005). Approximately 47 genes have been classified as putative AGPencoding genes together with another 19 putative genes encoding extensin-like proteins (Schultz et al., 2002). AGPs have been classified into classical or nonclassical types, AG peptides of 10- to 15-amino acid residues, and fasciclin-like AGPs (FLAs; Gaspar et al., 2001; Schultz et al., 2002). Classical AGPs are defined by the core protein containing Hyp, Ala, Ser, Thr, and Gly as the major amino acid constituents and the C terminus is glycosylphosphatidylinositol anchored. In nonclassical AGPs, other amino acids can be present and grouped into domains such as a Cys-rich or Asnrich domain. FLAs are chimeric AGPs that contain both an AGP motif and a fasciclin domain. In Arabidopsis, there are 14 classical AGPs, 12 AG peptides, an unknown number of nonclassical AGPs, and 21 FLAs (Borner et al., 2002, 2003; Schultz et al., 2002). 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, 1965a, 1965b; Schultz et al., 1998; Majewska-Sawka and Nothnagel, 2000; Gaspar et al., 2001; Showalter, 2001; Hall and Cannon, 2002). Although the precise roles of most individual AGPs remain unclear, biological activities have been identified for some AGPs, such as xylogen, a protein with an AGP-like region. It was proposed that the glycan moiety of xylogen is necessary for mediating local and inductive cell-to-cell interactions required for xylem differentiation (Motose et al., 2004). Other AGPs have been implicated in female gametogenesis

Plant Physiology, October 2006, Vol. 142, pp. 458–470, www.plantphysiol.org Ó 2006 American Society of Plant Biologists

Synthetic Proteoglycans in Arabidopsis

(AtAGP18), Agrobacterium binding (AtAGP17), abscisic acid signaling during seed germination (AtAGP30), and cell-to-cell adhesion (AtFLA4; Nam et al., 1999; Shi et al., 2003; van Hengel and Roberts, 2003; AcostaGarcia and Vielle-Calzada, 2004; Gaspar et al., 2004; van Hengel et al., 2004). On the other hand, extensins have been proposed to (1) contribute to wall architecture; (2) increase tensile strength; (3) participate in cell plate formation; and (4) contribute to defense responses by forming interpenetrating cross-linked networks in the wall, possibly through diisodityrosine and pulcherosine (Brady et al., 1998) intermolecular cross-links (Held et al., 2004). However, there have not been direct tests of the biological significance of these proposed functions. 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, 2001; Tan et al., 2003, 2004; Held et al., 2004). In most previous experiments, the synthetic glycopeptides were expressed in tobacco (Nicotiana tabacum) cells. In this work, expression of 10 different oligopeptides containing AGP and extension motifs as EGFP fusions was performed in stably transformed Arabidopsis plants. This allowed an analysis of (1) posttranslational modifications of specific HRGP motifs in Arabidopsis; (2) expression pattern of the motifs in vivo and in vitro during plant development; and (3) subcellular localization in different cell types. These experiments revealed that the expression of these molecules in Arabidopsis is a complex process involving posttranscriptional and developmental controls.

RESULTS Expression of SynGMs in Arabidopsis

Ten synthetic gene constructs based on two different types of Hyp-rich glycomodules (Shpak et al., 2001; Zhao et al., 2002; Tan et al., 2003) were expressed in stably transformed Arabidopsis plants under the control of the strong constitutive 35S promoter (Fig. 1). We refer to these proteins collectively as SynGMs (glycomodules expressed via synthetic genes) and the individual proteins are referred to by the repeating element (e.g. Ser-Pro-Pro-Pro repeats [SPPP]) or the parent AGP (e.g. AGP 1 from tomato [Lycopersicon esculentum; Le-AGP1]). One type of SynGM contained AGP motifs Plant Physiol. Vol. 142, 2006

Figure 1. Modular domain organization for the glycomodules expressed via synthetic genes (SynGMs; A–K) expressed under the control of the 35S promoter in Arabidopsis plants. SStom, LeAGP1 signal sequence; SStob, extensin signal sequence from tobacco. The drawings are not to scale. Th, Thr; (YK)20, P3 type of extensin repeat (SOOOOSOSOOOOYYYK)20; (GAGP)3, gum arabic glycoprotein repeat derived from Acacia GAGP; GPI, glycosylphosphatidylinositol.

[Ala-Pro repeats (AP)51, Val-Pro repeats (VP)11, Ser-Pro repeats (SP)32, and Thr-Pro repeats (TP)101] with an N-terminal tomato signal sequence and EGFP at the C terminus. A second type was based on extensin motifs [(SPP)24, (SPPP)15, (SPPPP)18, and (extensin of P3 type [YK])20] with a tobacco signal sequence. A third group based on AGPs present in Acacia and tomato plants contained both AGP and extensin motifs (gum Arabic glycoprotein [GAGP] and LeAGP1, respectively) under control of the 35S promoter (Fig. 1). An 11th construct was the control, EGFP fused to the tobacco signal sequence. 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 80 459

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Figure 2. SynGM expression in adult plant tissues. A, Western blots of SynGMs in extracts from roots (R), leaves (L), stems (St), flowers (F), and siliques (Si) probed with anti-GFP antibody. The expected relative molecular mass of each mature SynGM in the absence of glycosylation is indicated by the black arrowhead. The Mr of some of the SynGMs previously observed in tobacco cells (solid line), tobacco plants (broken line), and tomato plants (thin line) is indicated on the right side of sections. B, Effect of heat and urea on Mr of the SPPP fusion protein. Incubation of a leaf extract at room temperature (RT) with or without urea, before electrophoresis produced a band of expected molecular mass of the nonglycosylated construct (white arrowhead) together with other bands of higher Mr, but no band is detected at 27 kD (*). Samples incubated for 5 min at temperatures above 50°C resulted in a band at 27 kD corresponding to the EGFP protein that is cleaved from the rest of the glycoprotein (black arrowhead).

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 460

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), but the same molecular mass range when expressed in tobacco cells (Sun et al., 2004). LeAGP1 was present in all Arabidopsis tissues analyzed not only by western blots, but also by confocal laser-scanning microscopy (CLSM; Figs. 3A and 4, B and C). Similar tissue expression was found in tomato plants (Gao and Showalter, 2000). LeAGP1 orthologs in Arabidopsis (AtAGP17, AtAGP18, and AtAGP19) are Plant Physiol. Vol. 142, 2006


Table I. SynGMs expressed in Arabidopsis –, Not detected. SynGM Mr in Calculated BY2 Cells Molecular (Tob) and Tomato Massa in Plants (Tom) Arabidopsis

Observed SynGM Molecular Mass in Arabidopsis Plants (Detected by Western Blot)

Glycan Molecular Massb,c

SynGM Molecular Massc,d

kD

kD

kD

33.7 (Tob)e 105 (Tob)e,f

29.1 32.9

Approximately 17.7 Approximately 30.5

46.8 62.7

(TP)101 119 (Tob)e Extensin motif (SPPP)15 63–77 (Tob)h (SPPPP)18 66.4 (Tob)h (YK)20 152.3 (Tob)i AGP (GAGP)3 75 (Tob)f LeAGP1 90–200 (Tom)j; 164 (Tob)k

47.0

n.d.g

n.d.

32.7 35.5 62.1

Approximately 20.0 Approximately 21.0 Approximately 19.3

52.7 56.5 81.4

60–70; 90 80–90; 130 70–90

80–100 90; 130 60–75

65; 95 65; 95 90; 130 90; 130 68–70 60–70

31.4 42.3

n.d. Approximately 137.4

n.d. 189.1

40–55 150–200

45–55 130–200

– 50–125 65; 120–125 150–200 120–200 140–200

SynGM

kD

AGP motif (VP)11 (SP)32

Roots

Leaves

Stems

Flowers

Siliques

62–67 50–54

– 52–54

45–50

50–60

kD

48–50 45–50 – 37–40; 50–60; 37–60; 95–110 52–54 95–110 50 65–85 –

75–95 95 –

a b Based on DNA sequence, but not including signal sequence or praline hydroxylation. Based on the carbohydrate and protein content on the purified SynGM measured by phenol-sulfuric and bicinchoninic acid methods. In parentheses, the estimated glycan counterpart in the c d corresponding SynGM expressed in tobacco BY2 cells. SynGM purified from leaves. Molecular mass based on carbohydrate and protein e f g h content of the purified SynGM. From Tan et al. (2003). From Shpak et al. (1999). n.d., Not determined. From Shpak et al. i j k (2001). From Held et al. (2004). From Gao et al. (1999). From Sun et al. (2004).

transcribed in all tissues (Sun et al., 2005). Therefore, it is not surprising that LeAGP1 has the most normal expression and presumably hydroxylation/glycosylation of all the SynGMs tested. 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 pro-

tein 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

Table II. Pro hydroxylation and monosaccharide composition of the SynGMs expressed in Arabidopsis tr., Percentages lower than 1% are given as trace. Pro Hydroxylationa

SynGM

Monosaccharide Composition of the O-Glycans Linked to the SynGMsb Gal

Ara

%

AGPv motif (VP)11 (VP)11 (Tob)d (SP)32 (SP)32 (Tob)e Extensin motif (SPPP)15 (SPPP)15 (Tob)f (SPPPP)18 (SPPPP)18 (Tob)h (YK)20 (YK)20 (Tob)i AGP LeAGP1 LeAGP1 (Tob)j,k

Rha

GlcA

Gal:Ara (Molar Ratio)

mol %

59.0 (approximately 7 Hyp) 79.5 (approximately 9 Hyp) 95.3 (approximately 31 Hyp) 100 (approximately 32 Hyp)

34.7 40 (46) 57.7 45 (55.0)

38.3 39.0 (44.8) 30.1 28 (34.5)

26.9 8.0 (9.2) 12.2 8.0 (9.9)

n.d.c 13 n.d. 19

1:1.10 1:0.97 1:0.52 1:0.62

71.0 (approximately 35 Hyp) 73.3 (approximately 36 Hyp) 51.3 (approximately 37 Hyp) 97.5 (approximately 72 Hyp) 61.1 (approximately 110 Hyp) 100 (approximately 180 Hyp)

111g 37 (41.0) 111 5 57.2 2.8

11 42 (46.6) 11 95 29.0 90

6 7.0 (7.7) 6 – 13.8 1

n.d. 10 n.d. tr. n.d. –

n.d. 1:1.13 n.d. 1:19.0 1:0.51 1:11.2

94.7 (approximately 44 Hyp) 92.3 (approximately 43 Hyp)

68.9 37 (42)

18.4 39 (44.3)

12.7 8 (9.0)

n.d. 12

1:0.27 1:1.05

a

Based on the amino acid composition of the purified SynGM-EGFP fusions. In parentheses, the total number of hydroxyproline units per construct b c is shown. Monosaccharide composition before and after discounting the uronic acids (GlcA) for comparison. n.d., Not determined. d e f g i From Tan et al. (2003). From Shpak et al. (1999). From Shpak et al. (2001). 111, Abundant; 11, intermediate; 6, scarce. From j k Held et al. (2004). From Zhao et al. (2002). Four percent of mannose was also detected. Plant Physiol. Vol. 142, 2006

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Figure 3. Expression of SynGMs in seedlings. Pseudo green indicates fluorescence of EGFP-tagged SynGMs; pseudo red indicates fluorescence of PI staining from the cell walls and autofluorescence of the chlorophyll. Transmitted light (left) and confocal images (right) of 5-d-old seedlings. EGFP fluorescence is evident in roots, but not in most areas of shoots of SynGM-expressing transgenic plants. Scale bar 5 0.5 mm.

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. Biochemical Characterization of Purified SynGMs

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; Zhao et al., 2002; Tan et al., 2003). In contrast, the motifs (SPPPP)18 and (YK)20 that were completely hydroxylated when expressed in BY2 cells had much less Hyp (51.3% and 61.1%) when expressed in Arabidopsis leaves (Table II), suggesting that selective expression of different P4Hs but also dissimilar substrate specificity could be involved. 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

Figure 4. SynGM expression at protein and mRNA levels in seedlings. A, Western blots of proteins extracted from roots (R) and hypocotyls, cotyledons, and first leaves (H 1 L) of 5-d-old seedlings probed with anti-GFP antibody. Absence of GFP-SynGMs is indicated with asterisks. B, RT-PCR of EGFP or actin1 (25 and 35 cycles, respectively), using RNA from hypocotyls, cotyledons, and first leaves. Control (2RNA), Negative control without the addition of RNA; control (2RT), negative control without the addition of reverse transcriptase; EGFP (1control), positive control based on the pEZSLC plasmid containing the EGFP sequence.

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poor mobility of the highly glycosylated peptides in SDS gels due to the steric hindrance of SDS binding to the polypeptide backbone (Hames, 1990). The SynGMs monosaccharide composition (Table II) showed, as expected, that Gal, Ara, and Rha are the major sugars for the AGP motifs (VP)11, (SP)32, and LeAGP1. Somewhat surprisingly, similar sugar composition was observed for the extensin motifs (SPPP)15, (SPPPP)18, and (YK)20 SynGMs. The carbohydrate composition for the SynGMs with AGP motifs [(VP)11, (SP)32] was similar to that reported in tobacco cells (Shpak et al., 1999; Tan et al., 2003), although (VP)11 showed much higher Rha content, and LeAGP1 had higher Gal and Rha content in the glycan moiety (Table II). Although sugar composition was analyzed only in the purified SynGM obtained from leaves, these glycans with similar sugar ratios seem to be present in all the tissues as shown by their similar relative molecular mass in the western blots for (SPPP)15, (SPPPP)18, and LeAGP1 (Fig. 2). On the other hand, altered types of carbohydrate ratios in the glycans are expected for the rest of the SynGMs [(VP)11, (SP)32, (TP)101, (YK)20, and (GAGP)3] in the different plant tissues. CLSM of SynGMs in Seedlings

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. Plant Physiol. Vol. 142, 2006

Expression of SynGMs in Adult Plants

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 tissuespecific 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). Subcellular Localization of SynGMs

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 463

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Figure 5. Expression of SynGMs in adult plant. Confocal images of leaf (L) and stem (St) cross sections. Scale bar 5 100 mm. Pseudo green fluorescence is derived from EGFP-tagged SynGMs; pseudo red fluorescence is from chlorophyll autoflourescence and PI staining.

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; Sun et al., 2004) and not localized, as expected, in the ER endomembrane system (De Loose et al., 1991). In contrast, the intact EGFP 464

without the signal sequence is located in the ER in tobacco cells (Matsui et al., 2003). In all of the transgenic lines studied, no Hechtian strands were detected in roots or in leaves even at lower mannitol concentrations (at 100–200 mM) to induce a lower rate of plasmolysis. It is possible that during plasmolysis the Hechtian strand could have broken easily because it was observed in tobacco cells (Sun et al., 2004) and Ginkgo biloba callus cells (Buer et al., 2000). 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 Plant Physiol. Vol. 142, 2006


Figure 6. (Legend appears on following page.) Plant Physiol. Vol. 142, 2006

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[(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.

DISCUSSION

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, 2001; Zhao et al., 2002; Tan et al., 2003, 2004; Held et al., 2004). Two of the SynGM constructs that resulted in accumulation of SynGMs in tobacco, (AP)51, and (SPP)24, did not give any detectable protein in Arabidopsis for unknown reasons even if these repeats are also present in the backbone of LeAGP1. Possibly, these repeats were not recognized by some component of the Arabidopsis glycosylation process and were degraded; however, the AGP motifs (APAP)n and (SPP)n repeats are common in Arabidopsis AGPs, including LeAGP-1 orthologs AGP17 (At2g23130), AGP18 (At4g37450), and AGP19 (At1g68725). The other eight constructs gave rise to highly glycosylated proteins in Arabidopsis. The major posttranslational modifications observed for the SynGMs in Arabidopsis were the hydroxylation of Pro

Figure 7. Confocal images of subcellular localization of SynGMs in roots and leaves. Pseudo green indicates the fluorescence of EGFPtagged SynGMs; pseudo red indicates the fluorescence of PI staining from the cell walls. A to G, Roots cells. H and I, Abaxial epidermal cells from leaves. A, Soluble EGFP used as a control. Scale bar 5 10 mm. B and C, SP fusion protein is retained in the ER and accumulated in fusiform bodies (yellow arrowheads). Scale bar 5 10 mm. C, Connection between the fusiform bodies and the ER network (white arrowhead). Scale bar 5 10 mm. D and E, Plants expressing SPPP and SPPPP proteins had relatively few fusiform bodies (yellow arrowheads) in the cytoplasm. Scale bar 5 10 mm. F and G, Comparison between the nonsecreted SP and the secreted SPPPP in roots. Scale bar 5 10 mm. Fusiform bodies are indicated with yellow arrowheads. H, Secreted protein in the plasmolyzed epidermal cells in detail showing the location of the cell wall (CW, red arrowhead), plasma membrane (PM, white arrowhead), cytoplasm (asterisk), and the absence of fusiform bodies. Scale bar 5 10 mm. I, Partial three-dimensional reconstruction of an epidermal cell showing the EGFP signal in the ER network and close to the plasma membrane (no fusiform bodies were detected). Scale bar 5 2.5 mm. CW, Cell wall; PM, plasma membrane.

units and the addition of O-linked glycans on the synthetic peptides that represent from 35.5% (YK20) to 89.4% (w/w; VP11) of the mass of the glycoconjugate. The cell-type-specific pattern of posttranslational modifications presumably reflects both cell-typespecific expression and substrate specificity of P4Hs and glycosyltransferases. Furthermore, in some cases, two

Figure 6. Subcellular localization of SynGMs in untreated samples compared with samples in which plasmolysis was induced by 500 mM (A) or 800 mM (B) mannitol (treated). Pseudo green indicates the fluorescence of EGFP-tagged SynGMs; pseudo red indicates the fluorescence of PI staining. A, Abaxial epidermal cells from leaves in adult plants. Pseudo green indicates the EGFP fluorescence from control or EGFP-tagged SynGMs. Scale bar 5 15 mm. B, Differentiation zone (top) and root tip (bottom) of 5-dold seedlings. Scale bar 5 15 mm. Pseudo green indicates the fluorescence of EGFP-tagged SynGMs; pseudo red indicates the fluorescence of PI staining from the cell walls. 466



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; Tiainen et al., 2005), but also with the low hydroxylation of (SPPP)15 when expressed in tobacco cells, a rare motif in tobacco HRGPs (Shpak et al., 2001). On the other hand, the (SPPP) motif is common in Arabidopsis extensins where it is arabinosylated (K. Terneus and M. Kieliszewski, unpublished data; for an extensin isolated from Arabidopsis cell suspensions). According to the gene expression analysis database Genevestigator (Zimmermann et al., 2005), At-P4H1 and 2 are weakly expressed in leaves, whereas other putative At-P4Hs (such as At4g33910, At4g25600, and At5G66060) seem to be highly expressed in leaves and ubiquitously transcribed. Perhaps, in Arabidopsis leaves, which are generally Hyp poor, these three other putative P4Hs also have low affinity for the (SPPP)n, (SPPPP)n, and (YK)20 motifs. (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). PRPs show lots of clustered noncontiguous Hyp, but no AG addition, and it may be that the presence of abundant Y and K in the PRPs and extensins affects glycosylation of Hyp residues. Flanking sequence matters and clearly different species deal with noncontiguous Hyp in slightly different ways Plant Physiol. Vol. 142, 2006

being the most commonly conserved AG addition site for AOAO types in the AGPs and arabinosylation sites for SOOOO in the extensins. Lone instances of Hyp are arabinosylated, arabinogalactosylated, or remain nonglycosylated, depending on the flanking sequences. 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 celltype-specific expression of endogenous HRGP genes (Kieliszewski et al., 1992; Gao et al., 1999; Gao and Showalter, 2000), but also there appears to be cell-typespecific expression of factors involved in synthesis or degradation of HRGPs. All of the SynGMs used here (i.e. SPPP15, SPPPP18, and YK20) have previously been expressed in tobacco BY2 cells (Shpak et al., 1999, 2001; Zhao et al., 2002; Tan et al., 2003, 2004; Held et al., 2004) where they were efficiently modified and secreted (Shpak et al., 2001; Held et al., 2004) albeit at different levels. Similarly, all of the SynGMs were secreted in epidermal leaf cells of transgenic Arabidopsis. However, secretion of SynGMs in Arabidopsis roots varied from one construct to another. The (SPPP)15 and (SPPPP)18 constructs were at least partially secreted in the elongation zone of roots, whereas none of the other constructs were secreted. Where secretion was not observed, SynGMs were retained in the ER and accumulated in fusiform bodies. According to Gunning (1998), fusiform bodies are dilated cisterna of the ER found only in Brassicaceae (Iversen et al., 1983) and in other members of the Capparales (Behnke and Eschlbeck, 1978). Glycosylation type, signal sequence type, and/or the protein

Table III. Arabidopsis transgenic lines containing the SynGMs SynGM

Transgenic Lines

EGFP (control) (VP)11 (SP)32 (TP)101 (SPPP)15 (SPPPP)18 (YK)20 (GAGP)3 LeAGP1

SG0, SG1 SG179, SG180, SG182 SG60, SG65, SG75 SG4, SG5 SG114, SG118 SG125, SG145 SG188, SG189 SG198, SG199 SG6, SG163 467

Este´vez et al.

sequence (i.e. glycosylation sites) could be altering transit through the ER compartment and the final secretion to the plasma membrane in these cells. Because, in most cases, the amount of glycan mass attached to the various proteins was similar in roots and leaves, the implication seems to be that there are significant differences in the secretory pathway in leaves and roots. Because roots did not appear to secrete certain SynGMs [(VP)11, (SP)32, (YK)20, (GAGP)3], whereas others [(SPPP)15, (SPPPP)18, and LeAGP1] were only poorly secreted, we think it unlikely that the root secretory system was overwhelmed by the mass of SynGMs produced. Rather, we infer that the root secretory apparatus is unable to process certain types of sequences and, therefore, these proteins become trapped at an early stage of the secretory pathway. The observation that many of the SynGMs accumulate in the ER, rather than the Golgi, especially in root cells, and the fact that they also showed high Mr as a consequence of posttranslational modifications, may suggest that the ER is the site of O-linked glycosylation for HRGPs in plants. The accumulation of fusiform bodies may indicate that the SynGMs have initiated abnormally high levels of protein turnover in the ER (Hayashi et al., 2001). Glycosylation type, signal sequence type, and/ or the protein sequence (i.e. glycosylation sites) could be altering the transit through the ER compartment and the final secretion to the plasma membrane in these cells. Also, it is possible that constitutive expression of the SynGMs might overload the ER in the root cells. In leaf epidermal cells, even when there was overexpression of the glycoproteins, no fusiform bodies were detected in the ER in all the lines studied (Figs. 6B and 7H). Thus, the conditions necessary for induction of fusiform bodies are different in root cells compared with epidermal cells in leaves. 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. Este´vez 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) is clearly differentiated into two groups: (1) high transcription levels in a very tissuespecific manner comprising roots (At2g17720 and P4H-2), pollen (At4g35820 and AT5G18900), and callus and cell suspension (At1g20270 and At3g28480); and (2) low/intermediate expression levels, but ubiquitously transcripted in most of the plant tissues in many of the developmental stages. Also, P4H substrates like AGPs, FLAs, and extensins follow a similar expression scheme, telling us about probable different 468

P4H substrate specificities, but further characterization of P4Hs in vitro and in vivo are needed to have a better understanding of the HRGPs biosynthetic pathway. 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.

MATERIALS AND METHODS Plasmid Construction and Agrobacterium Transformation The plasmid constructions were reported elsewhere (Shpak et al., 2001; Tan et al., 2003) and the sequences of the BamHI-SstI fragments containing the various SynGMs are available at GenBank (accession nos. DQ389577-578 and DQ399411-413). Briefly, the entire signal sequence synthetic gene EGFP constructs were subcloned into the plant vector pBI121 as BamHI-SstI fragments in place of the b-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). The transgenic lines used here are listed in Table III. The SynGM (GAGP)3 sequence (SPSPTPTAPPGPHSPPPTL) is the repeat and is a variant of the real GAGP consensus repeat (SPSPTPTPPPGPHSPPPTL) because of an inadvertent A for P mutation.

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 m22 s21) 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.

RT-PCR 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 mg 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#).

Western-Blot Analysis Proteins were extracted using the method described in Martinez-Garcia et al. (1999). Briefly, plant material (two to three leaves, one stem, or 10 to 15 roots from seedlings at 5 dag, five siliques, 10 to 15 flowers) was ground in a microfuge tube in 300 mL 2 3 Laemmli buffer (125 mM Tris-Cl, pH. 6.8, 4% [w/v] SDS, 20% [v/v] glycerol, 2% [v/v] b-mercaptoethanol, 0.001% [w/v] bromphenol blue) containing 10 mL protease inhibitor cocktail (Sigma) and immediately transferred to ice. The samples (0.5–1.0 mg mL21 of protein) were boiled for 2 min and 20 to 30 mL were loaded on 10% SDS-PAGE. SynGM SPPP



and LeAGP1 protein extracts were treated using different conditions: (1) room temperature with or without 8 M urea (20 mL) in 20 mL of sample (RT urea and RT, respectively); (2) heated for 5 min at variable temperature from 50°C to 100°C without urea addition. In all cases, the proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Anti-GFP mouse IgG (clones 7.1 and 13.1; Roche Applied Science) was used at a dilution of 1:2,000 and it was visualized by incubation with goat anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (1:2,000) followed by a chemiluminescence reaction (Super-Signal; Pierce Chemical).

HRGP Extraction HRGP was purified according to Schultz et al. (2000) with modifications. Leaves were extracted with ethanol, chloroform-methanol (1:1), and finally with acetone until no pigments were visible and air dried. One to 5 g of precleaned freeze-dried tissue were ground to a fine powder in liquid nitrogen, added to 200 mL of extraction buffer (50 mM Tris-Cl, pH. 8, 10 mM EDTA, 1% Triton X-100, 0.1% b-mercaptoethanol), and incubated overnight at 4°C. Samples were centrifuged and the supernatant was precipitated in 5 volumes of ethanol (4°C, overnight). The pellet was extracted twice more in the same buffer (100 mL) and the supernatant obtained was precipitated as described above. The solvent-dried samples were dialyzed (molecular weight cutoff 6,000–8,000) in distilled water and freeze dried.

Light Microscopy Hand-cut stem cross sections (200 mm 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) was used to reference the tissues (leaves and stems) in the in situ localization of the SynGMs. Slides were viewed with a compound microscope (Leitz DMRB; Leica).

Supplemental Data 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 DEAESephacel anion-exchange chromatography.

ACKNOWLEDGMENT 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.

Isolation and Biochemical Analysis of the Oligopeptides 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 3 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). Protein content was determined using Bio-Rad protein assay based on the Bradford dye-binding procedure (Bradford, 1976) with bovine serum albumin as standard, and SynGMs were detected by immunodot assay using anti-EGFP monoclonal antibody as primary antibody already described for western blots. The EGFP fractions were collected, dialyzed, and freeze dried. An affinity column was prepared using 5 mL of the ImmunoPure protein G IgG Plus orientation kit (Pierce Biotechnology) to cross-link 400 mg of mouse antiEGFP monoclonal antibody (clones 7.1 and 13.1; Roche Applied Science) according to the manufacturer’s instructions. The EGFP positive fractions were solubilized in 1 mL of the binding buffer (phosphate-buffered saline 0.1 M, 0.15 M NaCl, pH 7.2) and applied into the affinity column. The samples were incubated for 2 to 3 h at room temperature and the column was washed with 20 volumes of the binding buffer. Bound proteins were eluted with 5 volumes of ImmunePure IgG elution buffer (0.1 M Gly-HCl, pH 2.8). Finally, the samples were desalted using a PD-10 column (Sephadex G-25 medium; Amersham-Pharmacia Biotech) and freeze dried. Molar protein and carbohydrate content on each SynGM were estimated colorimetrically as described before (using as an average molecular mass of 162 D for a sugar unit and 110 D for an amino acid residue). Based on the known molecular mass (without the signal sequence domain and not including the hydroxylation of the Pro residues), a preliminary estimation of the glycan molecular mass in each SynGM was carried out. Amino acid composition was determined at the Michigan State University macromolecular facility (East Lansing, MI).

CLSM and Imaging For in situ detection of the SynGMs on leaves and stem tissues, hand crosssections 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 60 3 Nikon PlanApo water immersion (WI) 1.2 numerical aperture (Technical Instruments), a 40 3 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.


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