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American Society of Plant Biologists The Arabidopsis SKU5 Gene Encodes an Extracellular Glycosyl Phosphatidylinositol–Anchored Glycoprotein Involved in Directional Root Growth
a Carnegie Institution, 260 Panama Street, Stanford, California 94305 1 To whom correspondence should be addressed. E-mail sedbrook{at}andrew2.stanford.edu; fax 650-325-6857
To investigate how roots respond to directional cues, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.
Roots grow by coordinating cell division and cell expansion at their tips. In Arabidopsis, this occurs within a 1-mm region of the tip composed of a meristem along with two zones of cell expansion termed the distal and main elongation zones (Mullen et al., 1998a  ). Within the meristem, cell division and differentiation begin with four sets of progenitor cells that surround the nondividing quiescent center cells (Dolan et al., 1993; Benfey and Scheres, 2000). Cells of each lineage divide to form concentric rings around the developing vasculature, first expanding quasiisodiametrically and then anisotropically as they pass through the elongation zones. Differentiating cells divide and expand anisotropically along the longitudinal axis of the root, forming distinct files (Dolan et al., 1993).
The cell files are in large part linear, although a slight twisting bias or handedness occurs naturally in roots of some Arabidopsis ecotypes, including Wassilewskija and Landsberg erecta (Mirza, 1987
As a root grows within a constant environment, cells of a given type divide and expand approximately the same amount around the periphery of the root, resulting in a straight root. Various stimuli induce a hormone-mediated differential growth process to occur, thereby altering the orientation of the root tip. For instance, a change in the root tip's alignment with the gravity vector induces an increase in cell expansion on the upper flank of the tip along with a decrease in expansion on the lower flank, causing the root tip to bend downward along a two-dimensional plane (Mullen et al., 1998a
More complex directional growth responses occur when roots are exposed to multiple stimuli. An example of this is the wavy root growth pattern. Okada and Shimura (1990)
Okada and Shimura (1990) To investigate the processes underlying regulated cell growth, we used root waving as a phenotype to facilitate the identification of mutants with alterations in growth processes. Here, we report the characterization of a novel mutant, sku5, which develops roots that skew away from the typical downward direction of growth on agar surfaces. sku5 roots and etiolated hypocotyls also twist more than normal in a counterclockwise direction about their longitudinal axes as they grow. Analyses of the growth response of the mutant shows that directional growth on agar surfaces can be uncoupled from axial root tip rotation, two processes that normally occur together. The SKU5 gene encodes a glycoprotein that is related structurally to the multiple-copper oxidases. SKU5 becomes glycosyl phosphatidylinositol (GPI) modified and localized to the plasma membrane and cell wall. Our results suggest that SKU5 plays an important role in regulating directional root growth.
Isolation and Characterization of sku5 The sku5 mutant was identified by screening a population of T-DNA–mutagenized seedlings for altered root growth on the surface of 1.5% agar-solidified germination medium (GM) that was tilted 30° from the vertical. Roots of wild-type seedlings grew in a waving pattern that was skewed slightly to the left when viewed from above the agar surface (Figure 1A , Table 1). The sku5 mutant was indistinguishable from the wild type except that the growth of mutant roots skewed strongly to the left and, in most cases, the roots looped to the left to form coils (Figure 1A).
When seedlings were grown on vertically oriented 0.8% agar-solidified GM, which did not impede vertical root growth, sku5 roots still slanted much more in a leftward direction than did the wild type (Table 1). On the other hand, when sku5 roots growing in a skewed direction on inclined agar medium penetrated the agar, they assumed a normal vertical trajectory of growth until they touched the bottom of the plate, after which they skewed again (Figure 1B). The growth directions of sku5 and wild-type roots that were grown for 7 days embedded in 0.8% agar-solidified GM also did not deviate significantly from the vertical (Table 1). These observations indicate that the sku5 skewed root growth phenotype was surface dependent and was not caused by a difference in the gravitropic set point angle.
Root waving has been associated with the formation of twisted epidermal cell files in places along the root where it has changed directions (Okada and Shimura, 1990 To determine if the sku5 counterclockwise cell file rotation bias also occurred in roots that were not subject to the directional stimuli found at a surface, we grew seedlings in 0.8% agar-solidified GM and in liquid GM, which presented the roots with minimal touch stimulation. Under these two conditions, sku5 roots twisted in a counterclockwise direction much more than wild-type roots, albeit slightly less than when grown on agar surfaces (Figures 1F and 1G, Table 1). The maximum angles at which the cell files crossed the longitudinal axes of the roots were approximately the same for both wild-type and sku5 roots. Under all of these conditions, the rate of epidermal cell file rotation varied along the lengths of the roots, with no apparent regularity. In some cases, both wild-type and sku5 roots rotated briefly in a clockwise direction about their axes. Cell file rotation was not seen in the first 400 µm of the root tip but became visible within the elongation zones (Figure 1G). This finding suggests that the axial twisting was caused by differential cell expansion that began in the elongation zones and was not the result of a skewed pattern of cell division in the meristem. Confocal microscopic imaging of propidium iodide–stained sku5 and wild-type roots revealed that in regions in which no cell file rotation occurred, epidermal and the underlying cortical cell files were parallel to each other (data not shown). In regions in which epidermal cell files were twisted, sku5 and wild-type cortical cell files also were twisted, albeit generally slightly less than the overlying epidermal cell files (Figures 2A and 2B) .
We also examined the hypocotyl cell files of sku5 and wild-type seedlings grown in the dark. We found that sku5 hypocotyls twisted more in a counterclockwise direction compared with the wild type (viewed along the axis toward the apex), indicating that the SKU5 gene also affects directional growth in this organ (average angles of cortical cells in relation to longitudinal axes: wild type, 3.8° ± 4.5°; sku5, 6.4° ± 5.1° [P = 0.001, 76 < n < 81]). We observed no obvious twisting or morphological defects in light-grown sku5 hypocotyls, petioles, and inflorescences (data not shown).
In addition to the surface-dependent directional growth defect of sku5 roots and the axial rotation defects of sku5 roots and etiolated hypocotyls, we found that sku5 roots and etiolated hypocotyls were To determine if sku5 roots were shorter than wild-type roots because the cells did not expand longitudinally to the same length as wild-type cells, we measured the lengths of cortical cells in the mature zones of sku5 and wild-type roots. Seedlings were grown for 7 days in 0.8% agar-solidified GM, and the roots were stained with propidium iodide and imaged with a confocal microscope. We found that sku5 cortical cells were the same length as wild-type cortical cells (sku5 average cell length was 203.5 ± 46.1 µm [n = 212], and wild-type average cell length was 200.4 ± 45.3 µm [n = 285]; P = 0.48 by Student's t test). We also observed no differences in the widths of cortical cells or other cell types within the root or the hypocotyl (data not shown). Cell sizes and shapes within the root tip, including the meristem and the elongation zones of sku5 roots, also appeared normal. We were unable to measure the lengths of sku5 epidermal cells because they twisted out of view.
Given that gravitropism plays an important role in root waving (Rutherford and Masson, 1996 We also germinated and grew sku5 and wild-type seedlings on 1.5% agar-solidified GM plates that slowly rotated in a clockwise direction on a clinostat. This treatment essentially randomized the seedling's exposure to the gravity vector, allowing observation of root growth character when this stimulus was minimized. After 5 days of growth, nearly all of the sku5 seedling roots had formed tight clockwise coils, whereas wild-type seedling roots had grown randomly or had taken on a slight clockwise growth direction (Figure 3) .
Although we did observe a "clinostat effect" in these experiments associated with the direction of seedling rotation (i.e., counterclockwise clinostat rotation tended to make roots grow in a counterclockwise direction [Mirza, 1988 ; Rutherford and Masson, 1996]), the results always showed that sku5 roots coiled much more in a clockwise direction than did wild-type roots (data not shown). Therefore, because wild-type roots did not coil like sku5 roots under randomized gravity, we conclude that an altered gravity response of sku5 roots is not the cause of the surface-dependent skewing phenotype. Rather, the gravitropic response counteracts the sku5 skewing phenotype.
We also examined how the microtubule-interacting drug propyzamide affected sku5 root growth. Furutani et al. (2000)
This treatment had the same effects on sku5 and wild-type Wassilewskija roots, except that the sku5 growth parameters were much more exaggerated than those seen in the wild type (data not shown). Additionally, propyzamide equally affected sku5 and wild-type root morphologies and lengths. Therefore, it appears that the sku5 mutant is not affected directly in the same processes as the spr mutants, which have defects in the orientations of their cortical microtubule arrays (Furutani et al., 2000
Cloning of the SKU5 Gene
An adaptor PCR method (Siebert et al., 1995
Fragment A also was used as a probe to screen a cDNA library (Kieber et al., 1993 ), resulting in the identification of four full-length or nearly full-length cDNA clones (pJS1 to pJS4). Sequence analysis of these clones, along with that of fragment B, revealed a 3.3-kb gene with nine exons encoding a putative 1764-bp open reading frame predicted to encode a 66-kD protein (Figure 4). The T-DNA had inserted within the fifth exon of that gene (Figure 4). To determine if the putative SKU5 gene could rescue the sku5 mutant phenotype, fragment B was cloned into the pBIB binary vector (Becker, 1990) to produce pJS5, which was introduced into sku5 plants via Agrobacterium tumefaciens–mediated transformation.
T1 transformants carrying this construct were isolated on hygromycin-containing Murashige and Skoog (1962) As a control, a 1.4-kb SacI-SmaI fragment within the putative SKU5 gene on pJS5 was removed, and the modified construct (pJS6) was transformed into sku5 plants. Phenotypic analysis of 15 T2 families from plants transformed with pJS6 revealed that all seedlings exhibited the skewed root-waving phenotype. Thus, we conclude that the gene carrying the T-DNA insertion was SKU5.
SKU5 Belongs to a Multigene Family That Is Related Structurally to the Multiple-Copper Oxidases
Two SKU5-like proteins from tobacco and tomato (NTP303, which has 45% amino acid sequence identity with SKU5 [Wittink et al., 2000
Database searches also revealed that the predicted SKU5 protein shares 23 to 27% amino acid sequence identity with ascorbate oxidases and laccases. These two families of multiple-copper oxidases coordinate four copper ions within three spectroscopically distinct centers (types 1, 2, and 3), allowing for the one-electron oxidation of a reducing substrate coupled to the four-electron reduction of oxygen to water (Messerschmidt and Huber, 1990 Searches of EST databases identified SKU5-related genes in at least 20 different monocotyledonous and dicotyledonous plant species (data not shown). SKU5 amino acid sequence alignments with the predicted protein portions encoded by these ESTs exhibited 51 to 86% amino acid sequence identity. This level of identity, along with the fact that they also do not contain all of the copper binding motifs, suggest that the genes corresponding to these ESTs are in the same family as SKU5 and the SKS genes.
SKU5 Is Expressed Ubiquitously
SKU5 Is Glycosylated SKU5 migrated more slowly than expected on SDS-PAGE gels (apparent mass of 90 kD instead of the predicted 66 kD; Figure 5A). To determine whether SKU5 was glycosylated, crude seedling extracts were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H), and the mobility of SKU5 was measured on protein gel blots of SDS-PAGE gels (Figure 6A) .
SKU5 within the PNGase F–treated sample migrated faster than that in the mock-treated sample, whereas SKU5 within the Endo H–treated sample migrated at the same rate as that in the mock-treated sample. We inferred that SKU5 was glycosylated with N-linked glycans that could be cleaved by PNGase F but not by Endo H. This pattern of differential glycanase susceptibility is symptomatic of proteins that pass through the Golgi, where the N-linked glycans become modified by a mannosidase, which makes them resistant to Endo H cleavage (Kornfeld and Kornfeld, 1985 ). Another line of evidence showing that SKU5 becomes modified post-translationally was obtained by in vitro translation of SKU5 in a reticulocyte lysate. This produced a protein that migrated on a SDS-PAGE gel at the predicted size of 66 kD (data not shown). When canine microsomal membranes capable of glycosylation were added to the system, the apparent mass of SKU5 increased to 90 kD (data not shown).
SKU5 Is Anchored by GPI
Three computer programs predicted that the SKU5 protein has the essential characteristics to become GPI modified: PSORT (http://psort.nibb.ac.jp/form.html; Nakai and Horton, 1999 We tested the possibility that SKU5 was anchored by GPI by determining whether the protein was released from membrane fractions by phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC cleaves the lipid component of the GPI anchor, solubilizing the protein. Microsomal membranes were fractionated by Triton X-114 phase partitioning to remove proteins that were associated peripherally with the membranes and then incubated with PI-PLC. The samples then were reextracted with Triton X-114, which resulted in the partitioning of the GPI anchor–released proteins into the aqueous phase. SKU5 was present in the PI-PLC–treated aqueous phase sample but not in the mock-treated control, confirming that SKU5 had been GPI anchored to membranes (Figure 6B).
SKU5 Localized to the Plasma Membrane and the Cell Wall
SKU5 was present in similar amounts in the 100,000g supernatant of wild-type extracts (Figure 7A, lane 1) and in the 100,000g pellet (lane 2). The amount of cytoplasmic contamination of the 100,000g pellet was low, as indicated by reprobing of the protein gel blot with an antibody against the cytoplasmic protein nitrilase (Figure 7B, lane 2). SKU5 also was detected in intracellular wash fluids extracted in a buffer with or without 1 M NaCl (Figure 7B, lanes 3 and 4). Thus, approximately half of the SKU5 protein is located in microsomal membranes, and the remainder is soluble and appears to be located in the cell wall. We also generated transgenic plants expressing a green fluorescent protein (GFP)::SKU5 fusion protein by placing the GFP gene in frame within the SKU5 gene of fragment B in a position just after the N-terminal leader peptide–encoding sequence (Figure 8A) . This construct was transformed into sku5 seedlings, in which it rescued the sku5 mutant phenotype (data not shown). Confocal microscopic analysis identified GFP fluorescence throughout the seedlings, with the brightest fluorescence occurring within the root meristem and the distal elongation zone (Figure 8B). This fluorescence could be seen completely surrounding cells of all tissue types, yet it appeared brighter along some parts of cells compared with others. Bright streaks also were seen along the longitudinal axis of the root, localized to three-cell junctions (Figure 8B).
These distinctive GFP-related fluorescence features did not occur with any consistent pattern. Faint GFP fluorescence also was visible within the cells; the illuminated structures appeared similar to those seen with endoplasmic reticulum– and Golgi-localized proteins (Figure 8C). With the filter set and gain settings used for these experiments, there was no detectable autofluorescence from nontransgenic plants (data not shown). To determine if any of the peripherally localized GFP fluorescence was associated with the plasma membrane, we treated roots with 100 mM mannitol to plasmolyze the cells. Confocal images of these roots showed that a considerable amount of fluorescence localized to the plasma membranes as they pulled away from the cell wall during plasmolysis (Figure 8D). Fluorescence also was associated with strands emanating from these membranes, which appeared to be Hechtian strands. Protein gel blot analysis of extracts from seedlings expressing the SKU5::GFP fusion protein, using either anti-SKU5 or anti-GFP antibody, identified a band of the predicted size of the fusion protein and bands of the sizes of SKU5 (detected by anti-SKU5 antibody) and the GFP protein (detected by anti-GFP antibody) (data not shown). Therefore, it appears that some of the fusion protein had been cleaved proteolytically, and consequently, some of the GFP-related fluorescence possibly did not reflect SKU5 localization accurately (data not shown). To clarify this issue, we isolated cellular fractions from SKU5::GFP-expressing transgenic seedlings and performed protein gel blot analysis. Our results showed that the anti-SKU5 antibody clearly detected the SKU5::GFP fusion protein in the microsomal membrane fraction from these preparations, whereas the anti-GFP antibody detected very little free GFP (data not shown). This finding indicates that the GFP-related fluorescence observed at the plasma membrane emanated predominantly (and perhaps exclusively) from the SKU5::GFP fusion protein. However, the anti-GFP antibody detected a large amount of free GFP in the cell wall fluid and soluble protein fractions, whereas the anti-SKU5 antibody detected relatively little SKU5::GFP fusion protein (data not shown). This finding suggests that the bright fluorescent streaks seen within the cell wall at the three-cell junctions probably came mostly from free GFP; hence, SKU5 may not localize normally to these spots at such high concentrations. We do not know why the three-cell junction fluorescence occurred only in distinct locations instead of uniformly.
Plants regulate root growth by controlling cell division and cellular expansion in the meristematic region and the elongation zones of the root tip. In doing so, they are able to respond to the numerous stimuli within their environment, avoiding those that are harmful and growing toward those that increase their likelihood of survival. Little is known about how plant roots sense multiple environmental stimuli and integrate the ensuing physiological signals into an optimal growth response. To dissect one aspect of root growth, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 with roots that responded abnormally to directional stimuli. The sku5 mutation was caused by a T-DNA insertion within the middle of the SKU5 coding sequence. RNA and protein gel blot analyses revealed that no mRNA or SKU5 protein was detectable in sku5 plants, so the mutation appears to be null. Phenotypic and molecular analyses of this mutant have provided novel insights into a previously uncharacterized process that appears to be required for normal root growth. When sku5 roots were grown in a homogeneous agar medium, they aligned normally with respect to the gravity vector. However, when grown on a vertical or tilted agar surface, sku5 roots consistently skewed to the left, away from the gravity vector at a characteristic angle. sku5 roots also skewed to the left when they were grown at the bottom of an agar plate against the plastic. This environment provided a differential touch stimulus but did not have the same directional moisture and ion gradients found at the surface. Thus, we infer that the surface-induced growth response is derived from a touch stimulus. sku5 roots grown on agar in the absence of a single gravity vector formed clockwise coils.
Therefore, it is likely that the growth angle of sku5 roots on the surface of inclined agar is maintained by an equilibrium between gravity-induced and surface-induced growth responses, not unlike that seen with the sku1 and sku2 mutants (Rutherford and Masson, 1996 The sku5 root tip axial rotation defect appeared constitutive, occurring while the roots were growing on an agar surface as well as in agar or liquid medium. This is in contrast to the directional growth phenotype, which appeared only when roots grew on an agar surface. The average rate of axial rotation, however, was slightly greater when the roots were grown on the agar surface, suggesting that a touch stimulus enhances the twisting. Most if not all mutants reported to have surface-dependent directional growth defects produce roots with an axial rotation defect.
In fact, mutants with roots that skew to the left exhibit counterclockwise axial rotations, whereas roots that skew to the right exhibit clockwise axial rotations (Mirza, 1987
sku5 roots were Only a few percent of the reduced root and hypocotyl length was attributable to the fact that roots composed of twisted cells are shorter than straight roots with the same number of cells. sku5 and wild-type root cortical cells are the same size. Therefore, it appears that sku5 roots were mostly shorter because of slower rates of cell expansion and cell division. Apparently, both expansion and division rates decreased proportionally, because we observed no differences in the sizes of sku5 meristems and elongation zones compared with those of the wild type (data not shown). It is unclear if the sku5 mutation directly affects cell division rates or if cell division is linked to a cell expansion defect. A sku5 effect on the rate of cell expansion seems likely because of the axial rotation defect, which is manifested in the elongation zones.
If cortical cells grew proportionally less than the overlying epidermal cells, then the epidermal cell files would have to twist to release the tension. Indeed, we observed that sku5 epidermal cell files twisted more than the underlying cortical cell files. This greater amount of epidermal cell file twisting also was observed in wild-type roots, suggesting that it was a naturally occurring phenomenon. Because the twisting almost always occurred in the same direction in sku5 roots, there must be an underlying directional growth bias. Furutani et al. (2000)
The SKU5 protein exhibits sequence similarity with the multiple-copper oxidases ascorbate oxidase and laccase. Ascorbate oxidases are cell wall–localized glycoproteins that catalyze the oxidation of ascorbic acid. This reaction appears to affect cell expansion, but the mechanism has not been determined (DeTullio et al., 1999
There has been much debate regarding whether laccases are involved in lignin polymerization (Ranocha et al., 1999 The Arabidopsis genome contains 18 other genes, provisionally designated SKS1 to SKS18, that are predicted to encode proteins with 44 to 65% amino acid sequence identity with SKU5. The predicted SKS proteins lack intact types 1, 2, and 3 copper centers. Therefore, these proteins probably do not possess oxidase activity unless they have evolved to bind copper in sites distinct from those seen in ascorbate oxidase and laccase. All of the predicted SKS proteins appear to have N-terminal leader sequences, like SKU5, while lacking any endoplasmic reticulum retention signals. This fact suggests that, like SKU5, they are secreted.
Orthologs of a few SKS proteins have been described previously as pollen-specific genes, including NTP303 from tobacco (Albani et al., 1992 Several lines of evidence indicate that SKU5 is GPI modified, N-glycosylated, and anchored to the plasma membrane. (1) An N-terminal peptide sequence in the Arabidopsis Plant Plasma Membrane Database corresponds to the site at which the SKU5 leader peptide is predicted to be cleaved. (2) PNGase F treatment showed that SKU5 is glycosylated. For glycosylation to occur, SKU5 must have been targeted to the endoplasmic reticulum. (3) Endo H treatment was unable to remove N-linked glycans, so the protein likely passed through the Golgi, where mannosidase made the glycans resistant to Endo H cleavage. (4) Detection of SKU5 in crude membrane preparations showed that it was membrane bound. (5) Plasma membrane localization was confirmed by the analysis of GFP-related fluorescence in seedlings expressing a SKU5::GFP fusion protein. (6) PI-PLC treatment released SKU5 from membranes, indicating that SKU5 had been anchored by GPI. (7) SKU5 was recovered in fluids extracted from the cell wall, suggesting that it was released from the GPI anchor in vivo, perhaps in a regulated manner.
The GPI modification of proteins has been found to occur in a variety of organisms, including animals, plants, fungi, and protozoa (McConville and Ferguson, 1993
Some GPI-anchored proteins become localized within the lumen of intracellular vesicles until they are secreted in a regulated manner (Lisanti et al., 1990 The analysis of transgenic seedlings expressing the SKU5::GFP fusion protein under the control of the SKU5 promoter showed that GFP-related fluorescence occurred all around cells in all cell layers of the root. When root cells were plasmolyzed with mannitol, some of the fluorescence localized to the plasma membrane. Although protein gel blot analysis of whole plant extracts showed that some of the SKU5::GFP fusion protein was cleaved to form free GFP and SKU5, protein gel blot analysis of microsomal membranes purified from the transgenic seedlings identified predominantly intact SKU5::GFP, with very little free GFP. Hence, it seems likely that the plasma membrane fluorescence originated from the fusion protein. The fluorescence appeared fairly uniform around the root cells, with only some random brighter patches of fluorescence occurring along cells in parts of the tip. Because these patches exhibited no obvious patterns, we conclude that the localization of SKU5 is not polarized. We envision a number of different models to explain SKU5 function. For instance, SKU5 might modify linkages between cell wall structural components, allowing for turgor-driven cell extension to occur in such a way to provide a directionality to cell expansion without affecting the cell's ability to achieve normal size. Because sku5 mutant roots twist, there must be an underlying growth imbalance that SKU5 activity normally counteracts. SKU5 activity also must counteract the surface-induced growth response for roots to grow in a normal downward direction. Alternatively, SKU5 may affect a property of the middle lamella that would allow cells to slide past each other as they grow. Because epidermal cells normally grow to be longer than the underlying cortical cells, a torsional strain will develop if the primary cell walls of these two cell types do not grow smoothly past each other. The strain could be released by the kind of twisting of the organ that is observed in the sku5 mutant. It also is conceivable that instead of modifying cell wall structural properties directly, SKU5 might act on a component in the cell wall that is in a signaling pathway, affecting directed cell expansion. Additional studies will be required to discern exactly what role SKU5 plays in controlling growth. Perhaps detailed structural studies will reveal an alteration in a component of the sku5 cell wall. This would facilitate the identification of potential substrates and enzymatic activity of the SKU5 protein. Along those lines, Fourier transform infrared spectroscopic analysis of plant tissues as well as quantitative analysis of neutral sugars derived from cell walls revealed no differences between sku5 and the wild type (data not shown). To examine the possibility of a regulatory function, comparison of gene expression in the mutant and the wild type using DNA microarrays may be useful. It also is possible that analysis of mutations in some of the SKS genes may result in a phenotype that is more suggestive of enzymatic function than is the sku5 mutation. The large collections of insertion mutants available in Arabidopsis should greatly facilitate the implementation of this approach.
Plant Stocks and Manipulation Wild-type Arabidopsis thaliana seeds of the ecotype Wassilewskija were provided by Tim Caspar (DuPont, Wilmington, DE). The sku5 mutant, ecotype Wassilewskija, was isolated from the Versailles collection of T-DNA insertion mutants (pGKB5 binary vector) (Bechtold et al., 1993 ; Bouchez et al., 1993). All techniques and conditions used to grow and manipulate Arabidopsis seeds, seedlings, and plants were as described previously (Rutherford and Masson, 1996).
Analysis and Quantification of Organ Growth
Confocal imaging of organs was performed with a Nikon Diaphot 200 inverted microscope equipped with a Nikon x40 1.15 or a Nikon x60 1.2 numerical aperture water immersion objective (Tokyo, Japan) and a Bio-Rad MRC 1024 confocal head with a krypton-argon laser. Enhanced green fluorescent protein (EGFP) was excited at 488 nm, and emitted fluorescence was collected through a 525/30 bandpass filter. Three-dimensional reconstructions of image stacks were generated with Lasersharp software (Bio-Rad) or NIH Image software. For confocal imaging, seedlings were immersed in half-strength Murashige and Skoog (1962)
Exogenous propyzamide (Chem Service, Inc.; www.chemservice.com/) treatments were performed as follows. Seeds were sown onto autoclaved 3MM paper strips that were positioned on 0.8% agar (type E; Sigma)-solidified germination medium (half-strength Murashige and Skoog [1962]
Cloning and Analysis of the SKU5 Gene and cDNAs
Generation and Analysis of Transgenic Plants Carrying a SKU5::Green Fluorescent Protein Reporter Construct
Generation of Anti-SKU5 Antibodies and Testing for SKU5 Glycosylation The anti-SKU5 antibody was used at a dilution of 1:1000, followed by 1:7500 dilution of a goat anti-rabbit IgG horseradish peroxidase–conjugated secondary antibody (Pierce). The Supersignal West Pico chemiluminescent substrate (Pierce) was used for horseradish peroxidase detection. To determine if SKU5 was glycosylated, 1-week-old seedlings were ground in the denaturing buffer supplied with the peptide N-glycosidase F and endoglycosidase H enzymes (New England Biolabs, Beverly, MA) and treated according to the manufacturer's instructions. The anti–protein disulfide isomerase antibody was purchased from Rose Biotechnology (www.rosebiotech.com/).
Fractionation of SKU5 and Glycosyl Phosphatidylinositol Anchor Determination
Cell wall fluid was isolated by vacuum infiltrating 7-day-old intact seedlings for 15 min with ice-cold buffer (100 mM Tris-HCl, pH 7.3, 5 mM EDTA, and 0.05%
The glycosyl phosphatidylinositol (GPI) modification of SKU5 was demonstrated by its removal from membrane fractions with phosphatidylinositol-specific phospholipase C, as described in Sherrier et al. (1999) The lower Triton X-114/membrane–containing phase was diluted 10-fold with 100 mM potassium phosphate buffer, pH 7.4, and divided into two equal parts; then, 1 unit/100 µL of phosphatidylinositol-specific phospholipase C (Sigma) was added to one part while an equal volume of 100 mM potassium phosphate buffer was added to the other part as a control. These samples were incubated for 1.5 h at 30°C with periodic mixing and then centrifuged at 1000g for 10 min. The aqueous upper phase was collected, 1 µg of BSA was added as an internal standard for protein recovery, and the samples were precipitated with 10% trichloroacetic acid (incubated on ice for 2 h and then centrifuged at full speed for 10 min in a microcentrifuge). The pellets were resuspended in SDS-PAGE loading buffer, the pH was neutralized with ammonium hydroxide fumes (an ammonium hydroxide–saturated Q-tip was held in the tube until the bromphenol blue turned from yellow to blue), and the pellets were electrophoresed on an SDS-PAGE gel and protein gel blotted as described above.
Accession Numbers
We thank Herman Höfte for providing the Versailles collection of T-DNA insertion mutants, the ABRC for the Texas A&M University BAC filters and clones as well as a SKU5 cDNA clone, Sean Cutler for the anti-nitrilase antibody, Seth Davis for the anti-GFP antibody, David Ehrhardt for assistance with confocal microscopy and for fruitful discussions, Paul Dupree for technical advice on demonstrating GPI anchoring, John Christie for technical advice, and Todd Richmond, Gert de Boer, Sean Cutler, Dario Bonetta, and other members of the Carnegie Institution for technical advice and fruitful discussions. This work was supported in part by grants from the Department of Energy to C.R.S. (Grant DOE-FG02-00ER20133) and from the National Aeronautics and Space Administration (Grants NAG2-1189 and NAG2-1492), the National Science Foundation (Grant MCB-9905675), and Wisconsin Hatch funds (WIS04310) to P.H.M. J.C.S. was supported in part by a National Institutes of Health postdoctoral fellowship (Grant 1 F32 GM20181).
Online version contains Web-only data.  Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002360. Received February 20, 2002; accepted April 3, 2002.
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Abstract
INTRODUCTION
15 and 10% shorter, respectively, than those of the wild type (
vector (Kieber et al., 1993
-mercaptoethanol), electrophoresed on SDS-PAGE gels, transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA), and analyzed using standard procedures. 
