In Vivo Colocalization of Xyloglucan Endotransglycosylase Activity and Its Donor Substrate in the Elongation Zone of Arabidopsis Roots

Correspondence to: Stephen C. Fry, S.Fry{at}Ed.Ac.UK (E-mail), 44-131-650-5392 (fax)

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO–SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO–SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide–SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO–SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The primary cell walls of flowering plants consist fundamentally of a framework of cellulose microfibrils embedded in a matrix of hemicellulose, pectins, and structural proteins (Carpita and Gibeaut 1993 ; Brett and Waldron 1996 ). Xyloglucan, the major hemicellulosic polysaccharide in the primary cell wall matrix of dicots, consists of a backbone of ß-(14)–linked D-glucose residues, the majority of which are -D-xylosylated at O-6. Some xylose residues are further substituted by galactosyl and fucosyl-galactosyl groups. Other, minor carbohydrate side chains and O-acetyl groups are also present (Fry 1989a ; Hayashi 1989 ). Because xyloglucans can form tight hydrogen bonds with cellulose microfibrils (Valent and Albersheim 1974 ; Hayashi et al. 1987 , Hayashi et al. 1994a , Hayashi et al. 1994b ; Hayashi 1989 ), they may thereby tether adjacent microfibrils (Fry 1989b ). A proportion of the xyloglucan molecules are covalently attached to acidic pectins (Thompson and Fry 2000 ). Xyloglucans also serve as storage polysaccharides in some seeds (Edwards et al. 1985 ).

For plant cells to expand, cellulose microfibrils in parallel alignment need to move apart or past one another, and this movement may create the possibility for newly synthesized xyloglucan molecules to become hydrogen-bonded (Fry 1989b ). Because xyloglucan tethers are thought to be the principal tension-bearing molecules in the cell wall, breaking of the tethers has been proposed as a mechanism for achieving reversible cell wall loosening in elongating tissue without compromising strength (Fry 1989b ; Hayashi 1989 ; Hoson et al. 1991 ). Although the cell wall contains numerous enzymes that can modify polysaccharides (Fry 1995 ), xyloglucan endotransglycosylases (XETs) seem well suited to play a predominant role in expansion. XET cleaves a xyloglucan chain (the donor substrate) endolytically and forms a covalent polysaccharide–enzyme complex (Sulova et al. 1998 ; Steele and Fry 1999 ); a new bond then forms between the new (potentially reducing) end and the free nonreducing end of another xyloglucan chain or of a suitable xyloglucan-derived oligosaccharide (XGO; the acceptor substrate) (Baydoun and Fry 1989 ; Smith and Fry 1991 ; Fry et al. 1992 ; Nishitani and Tominaga 1992 ; Lorences and Fry 1993 ).

Fry et al. 1992 hypothesized that XET-catalyzed transglycosylation reversibly loosens the cell wall, as is required for turgor-driven cell expansion, and some findings favor this hypothesis. XET activity is often correlated with growth rate (Fry et al. 1992 ; Hetherington and Fry 1993 ; Pritchard et al. 1993 ; Potter and Fry 1994 ; Xu et al. 1995 ; Palmer and Davies 1996 ; Antosiewicz et al. 1997 ; Catala et al. 1997 ). Xyloglucan turnover is correlated with auxin-induced elongation (Labavitch and Ray 1974 ; Nishitani and Masuda 1982 ), and in dicots, both auxin-induced elongation and xyloglucan breakdown are inhibited by lectins and by antibodies that bind xyloglucans and thereby presumably shield them from enzymic attack (Hoson and Masuda 1991 ; Hoson et al. 1991 ).

Potentially contradictory evidence, however, was obtained by McQueen-Mason et al. 1993 , who found that extracts containing active XETs from cucumber hypocotyls were unable to cause wall extension in hypocotyls in which the endogenous proteins had been denatured and that expansins (proteins that did induce extension in this system; McQueen-Mason et al. 1992 ) did not exhibit any measurable XET activity. Nevertheless, their work did not establish whether the exogenous XETs permeated the cell walls and catalyzed any transglycosylation reactions there. Although extractable XET activity exhibits a marked coincidence with the initiation of extension in maize roots and leaves, substantial activity could also be detected in mature tissue that was still turgid but had ceased extension (Pritchard et al. 1993 ; Palmer and Davies 1996 ). Thus, wall-tightening processes may be capable of overriding the wall-loosening effects of XET.

Besides the proposed role of XETs in cell wall loosening, these enzymes may also favor integration of newly synthesized xyloglucans into the cell wall (Xu et al. 1996 ; Nishitani 1997 ). Such integration is another necessary element for continued cell expansion. A role for XET in xyloglucan integration has been supported by the demonstration that newly secreted xyloglucan chains undergo interpolymeric transglycosylation at the time of their binding to the cell wall (Thompson et al. 1998 ).

Roles other than the two mentioned above can also be suggested. An enzyme with XET and xyloglucan endohydrolase activity is involved in the postgerminative mobilization of xyloglucan storage reserves in nasturtium (Tropaeolum majus) cotyledons (Farkas et al. 1992 ; Fanutti et al. 1993 ). XET activity increases during fruit ripening of kiwi (Redgwell and Fry 1993 ), tomato (Maclachlan and Brady 1994 ), and persimmon (Cutillas-Iturralde et al. 1994 ), suggesting a role in softening the cell wall and possibly in preparing it for further modification by other wall-associated (e.g., pectolytic) enzymes. Because Arabidopsis XETs accumulate in epidermal tissues under mechanical stress, XETs may contribute to the strengthening of cell walls to minimize any mechanically induced cell and tissue damage. They may also partially lyse walls to allow formation of xylem and phloem conducting elements and intercellular spaces (Saab and Sachs 1995 ; Antosiewicz et al. 1997 ).

Diverse factors regulate the expression of XETs and XET-related (XTR) proteins, including auxin (Xu et al. 1995 , Xu et al. 1996 ; Catala et al. 1997 ), abscisic acid (Wu et al. 1994 ), gibberellin (Potter and Fry 1993 ; Smith et al. 1996 ), ethylene (Redgwell and Fry 1993 ), and brassinosteroids (Zurek and Clouse 1994 ). Upregulation is also triggered by various environmental stimuli, including touch, darkness, temperature shock (Xu et al. 1995 , Xu et al. 1996 ), wind (Antosiewicz et al. 1997 ), and flooding (Saab and Sachs 1995 ).

To date, the best-documented but still hypothetical role of XET is its involvement in cell wall loosening during cell growth. Further evidence for or against this proposed role would be gained by in situ colocalization of XET activity and appropriate donor substrates. Several assays are currently available for extracted XET activity in vitro (Fry et al. 1992 ; Nishitani 1992 ; Lorences and Fry 1993 ; Sulova et al. 1995 ; Fry 1997 ), but in all these assays, an exogenous donor substrate is supplied, precluding any conclusions about substrate–enzyme colocalization. The method described by Fry 1997 is based on incorporating sulforhodamine-labeled oligosaccharides of xyloglucan (XGO–SRs), which act as acceptor substrates, into high-M_r products to yield a fluorescent polymer (xyloglucan–SR) that remains hydrogen-bonded to filter paper after the remaining unreacted XGO–SRs have been washed off. The method has been used for semiquantitative in vitro assays with "dot-blots," having been shown to work for tissue prints of celery petioles (Fry 1997 ), for example, and zymograms (Iannetta and Fry 1999 ). The method has now been adapted for in situ work, and here we report on the in vivo colocalization of XET activity and accessible donor substrate xyloglucans in various plant organs.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

In an initial experiment, transverse sections of celery petioles were assayed for simultaneous colocalization of XET activity and xyloglucan by using exogenous XGO–SRs. Bright orange fluorescence, resulting from the formation of wall-bound xyloglucan–SR, was typical of the collenchyma strands, the vascular bundles, and the epidermis, whereas the cortical cell walls displayed a fainter staining, as shown in Fig 1A. The image of the larger vascular bundle (Fig 1B) confirms the fluorescence in both xylem and phloem and the fainter staining in cell walls of cortical cells. In the upper and lower left-hand corners of Fig 1B, parts of two brightly stained collenchyma strands can be seen.

View larger version (74K): [in this window] [in a new window]   Figure 1. XET Activity–Donor-Substrate Colocalization in Celery Petioles and Arabidopsis Roots. (A) Celery petiole section showing high XET–donor colocalization, especially in collenchyma (c, collenchyma; e, epidermis; p, parenchyma; v, vascular bundle). (B) A larger vascular bundle showing bright fluorescent staining. (C), (D), (F), (G), (J), and (K) XET–donor colocalization in Arabidopsis roots. (C) Root from a 2-day-old Arabidopsis plant showing a distinct pattern of XET–donor colocalization coinciding with the elongation zone. (D) Bright-field image of the specimen shown in (C). Fig 1. (continued). (F) and (G) As in (C) and (D) except that the root is from a 5-day-old plant. (J) and (K) As in (C) and (D) except that the root is from a 13-day-old plant. (E) Close-up of the brightly fluorescent cell elongation zone near the root tip. (H) and (I) Young, emerging lateral roots. Fluorescence (H) and bright-field image (I). (L) and (M) An older lateral root, showing bright fluorescent staining of its elongation zone, as in the main roots. Fluorescence (L) and bright-field image (M). Bars = 100 µm.

Fig 1C depicts a 2-day-old Arabidopsis root, which displays a very distinct pattern of fluorescence. The root tip is almost devoid of fluorescence, the zone just behind the tip shows very bright fluorescence, and the more basal part of the root contains almost no detectable fluorescent staining except in the vascular tissue. The bright-field image of the same root helps to localize the zones shown in Fig 1C. A close-up of the bright zone near the tip, illustrated in Fig 1E, shows that the fluorescent cells increase in size going from the tip toward the base of the root (from left to right on the image), indicating that the area that is especially brightly stained is the cell elongation zone of the root.

Fluorescence and bright-field images of 5- and 13-day-old Arabidopsis roots are depicted in Fig 1F and Fig 1J, respectively. The size of the brightly fluorescent zone—the cell elongation zone of the root—increased with the age of the root. Fig 1H and Fig 1L illustrate the fluorescence pattern arising from XET–donor-substrate colocalization in lateral roots. In Fig 1H, a very young lateral root (at the left) exhibits no fluorescence, and an older one (at the right) shows some vague fluorescence at its base, where cells probably start to elongate. The epidermal cells of the main root show more intense fluorescence where the lateral roots emerge. In Fig 1L, the fluorescence pattern in an older lateral root resembles that in a young main root, with a distinct area of strong fluorescence in the elongation zone. Farther back along this lateral root, young root hairs are also seen to be associated with intense fluorescence. In general, the root hair cell walls were uniformly stained.

To check whether this specific labeling pattern was more general, roots of Arabidopsis, grown in soil under greenhouse conditions, and tobacco roots were assayed. The Arabidopsis root in Fig 2A shows a labeling pattern consistent with that seen in roots grown in vitro. In tobacco (Fig 2G), the fluorescence pattern and intensity are comparable to those of the Arabidopsis roots (Fig 1C, Fig 1F, and Fig 1J), although the elongation zone is longer.

View larger version (62K): [in this window] [in a new window]   Figure 2. XET Activity–Donor-Substrate Colocalization in Arabidopsis and Tobacco Roots. (A) and (B) Root of a 5-day-old Arabidopsis plant grown in soil and assayed for XET–donor colocalization (A); the corresponding bright-field image is shown in (B). (C) and (D) Controls showing lack of XET activity in a boiled root. Fluorescence (C) and bright-field image (D). (E) and (F) Control showing assay with cellotetraose–SR as the potential acceptor substrate. Fluorescence (E) and bright-field image (F). (G) Tobacco root assayed for XET–donor colocalization (fluorescence). Bars in (A), (C), and (E) = 100 µm; bar in (G) = 300 µm.

To verify that XET activity was being assayed, we included several controls in our measurements. Roots showed no autofluorescence in response to green light excitation after incubation in Mes buffer that lacked XGO–SR. Fig 2C illustrates the results for an Arabidopsis root assayed after it had been boiled for 2 min in water. The structure of the root can be seen in the bright-field image, whereas the fluorescence image shows that boiling resulted in the total loss of ability to yield any fluorescence detectable with the same manipulations and camera settings used for experimental roots that had not been boiled.

When Arabidopsis roots were incubated with cellotetraose–SR instead of XGO–SR, little or no fluorescent product was discernible. (A specimen in which a small amount of fluorescence was detected is shown in Fig 2E.) Incubation with cellobiose–SR gave similar results. Compared with the normal fluorescence pattern (Fig 1C, Fig 1F, Fig 1J, and Fig 1L), the elongation zone of the roots was not appreciably stained. Instead, a vague signal was generated in a zone nearer to the root tip.

When Arabidopsis and tobacco roots were assayed with XGO–SR solution supplemented with 0.08 or 1 mM unlabeled XGOs, fluorescence drastically decreased for both XGO concentrations (results not shown).

Arabidopsis and tobacco roots were incubated with XGO–SR in 25 mM Mes buffer at various pH values. In roots incubated at pH 4.0 (results not shown), fluorescence intensity was much less than in roots treated at the usual pH of 5.5 (Fig 1C, Fig 1F, and Fig 1J for Arabidopsis; Fig 2G for tobacco). Comparison of fluorescence intensities in roots incubated at pH 5.5 and 7.0 showed no appreciable difference (results not shown).

Fig 3 shows that the thin-layer chromatographic (TLC) profile of XGO–SRs reextracted from tobacco roots after a 1-hr incubation (Fig 3, lane 2) was similar to that of the starting material (Fig 3, lane 3), indicating that little hydrolysis of the fluorescently labeled substrate had occurred during the assay. Lanes 4 to 8 establish the relationship between chromatographic mobility and molecular mass for a range of oligosaccharide–SR conjugates. A trace of glucose–SR was present in most preparations, probably indicating a slight nonenzymic hydrolysis of the terminal glycosidic linkage. XGO–SRs reextracted from Arabidopsis roots (Fig 3, lane 1) showed evidence of partial conversion of the nonasaccharide (XLLG–SR) to octasaccharide(s) (probably XXLG–SR or XLXG–SR [or both]) and smaller products, indicating some glycosidase activity; however, throughout the 1-hr incubation, the majority of the XGO–SRs remained large enough to be acceptor substrates for XET.

View larger version (106K): [in this window] [in a new window]   Figure 3. TLC of XGO–SRs before and after Incubation with Arabidopsis and Tobacco Roots. After incubation in the presence of the roots for 1 hr, remaining XGO–SRs were reeextracted and analyzed by TLC. This image is a negative (dark spots indicate fluorescence). Lane 1 contains XGO–SRs reextracted from Arabidopsis root; lane 2, XGO–SRs reextracted from tobacco root; lane 3, XGO–SR mixture initially supplied to the roots; lanes 4 to 6, XGO–SRs (including XLLG–SR [DP9], XXLG–SR [DP8], XXXG–SR [DP7], XXG–SR [DP5], XG–SR [DP3], and glucose–SR [DP1]); lane 7, a mixture of cello-oligosaccharide–SRs spanning DP4 (cellotetraose–SR) to DP1 (glucose–SR); lane 8, a mixture of malto-oligosaccharide–SRs spanning DP6 (maltohexaose–SR) to DP1 (glucose–SR). Numbers to the left of major spots indicate the degree of polymerization of the oligosaccharide moiety. In each case, the former reducing group of the carbohydrate is a 1-amino-1-deoxy-D-glucitol moiety, to which sulforhodamine is attached by a sulfonamide bond.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Most studies of XET expression in relation to cell expansion have used (1) measurement of extractable enzyme activity assayed in vitro (Fry et al. 1992 ; Hetherington and Fry 1993 ; Pritchard et al. 1993 ; Potter and Fry 1994 ; Xu et al. 1995 ; Palmer and Davies 1996 ); (2) immunocytochemistry with antibodies against the XET protein (Antosiewicz et al. 1997 ); or (3) assessment of XET mRNA concentrations (Zurek and Clouse 1994 ; Xu et al. 1995 , Xu et al. 1996 ; Catala et al. 1997 ; Nishitani 1997 ). None of these approaches is ideal. Method 1 has limited spatial resolution and may fail to extract all the active XET from the tissue; in particular, covalent polysaccharide–enzyme complexes (Sulova et al. 1998 ; Steele and Fry 1999 ) may not be eluted from the cell wall. Method 1 may also extract any intraprotoplasmic XETs, which would not be capable of acting on wall xyloglucans in vivo because of compartmentalization. Method 2 detects XETs and related proteins regardless of whether they possess enzymic activity. Method 3 takes no account of the possible translational or post-translational control of XET expression.

A fourth approach, reported here, is the in situ colocalization of XET activity and accessible donor substrates. The fluorescent acceptor substrates used (XGO–SRs) are relatively large (~1.6 to 2.0 kD), hydrophilic molecules that are not taken up by living protoplasts; in vivo, therefore, the method detects only extraprotoplasmic XETs, that is, those enzyme molecules that potentially have access to substrates in the cell wall. Furthermore, because our assay relies on endogenous extraprotoplasmic xyloglucan as the donor substrate, it cannot give false positives attributable to the presence in the cell wall of active XETs that are incorrectly located to come into contact with a suitable donor substrate. In addition, our method clearly detects only active XETs, not inactive proenzymes or other structurally related proteins that lack transglycosylase activity. Finally, unlike method 3, it cannot give false positives caused by the failure to take into account the possible regulation of XET gene expression at the translational or post-translational level.

The product of the assay, xyloglucan–SR, could be either an integral cell wall component or a compound that is soluble in the apoplast. Our first washing solvent (ethanol/formic acid/water, 15:1:4 [v/v/v]) in which XGO–SRs are soluble but polysaccharides are insoluble would retain both types of product in situ; the second solvent (5% formic acid) would remove any soluble apoplastic xyloglucan–SR and leave only those products that were firmly wall bound. In the present work, we restricted our observations to the latter.

In fresh sections of celery petioles, the strong XET–donor-substrate colocalization seen in the collenchyma, vascular bundles, and epidermis in comparison with that in cortical cells confirms and adds spatial resolution to tissue print patterns obtained by Fry 1997 . The collenchyma is composed of thick-walled cells that provide mechanical support to the petiole but are nevertheless capable of growth in length. It seems reasonable to suggest that elongation of the thick collenchyma cell walls requires particularly high quantities of wall-loosening activities such as that of XET.

A very characteristic distribution pattern of XET–donor-substrate colocalization was found in Arabidopsis and tobacco roots. In both species, strong fluorescence is limited to the cell elongation zone of the root. Other regions exhibit very low fluorescence, supporting the correlation between XET action and cell extension. This correlation is further highlighted since the strong fluorescence signal, mainly restricted to the zone of cell elongation, increased steadily during the ageing of the roots as does the growth zone of the roots (Beemster and Baskin 1998 ). Lateral roots also mirrored this behavior.

Several controls provided evidence that the formation of the fluorescent product was the result of XET activity rather than a physical artifact or the action of different wall enzymes (Fry 1995 ). The loss of activity after boiling is an indication that the fluorescent signals obtained in experimental roots were the result of enzymic activity and not adsorption of the substrate. The failure of other fluorescent substrates such as cellotetraose–SR (chemically identical to the backbone of XGO–SR but lacking the nonreducing terminal xylose residue that is essential for recognition as an acceptor substrate by XET [ Lorences and Fry 1993 ]) and cellobiose–SR to give strong fluorescence in the zone of cell elongation indicates that the activity is specific for XET. Competition experiments with unlabeled XGOs confirmed the specificity of the enzymic activity. XETs have a K_m for XGOs of ~20 to 80 µM (Fry et al. 1992 ; Purugganan et al. 1997 ); therefore, XGOs added at or above this concentration would be expected to compete with the XGO–SRs and diminish the XET-catalyzed production of xyloglucan–SR, as we observed.

Analysis by TLC of the nonincorporated XGO–SRs that remained showed that ß-galactosidase activity in the wall was only slight. If present, high ß-galactosidase activity, followed by the action of -xylosidase and ß-glucosidase (Koyama et al. 1983 ), would have digested the XGO–SRs, eventually forming XET-inactive products such as the trisaccharide Xyl-Glc-Glc–SR (XG–SR) (Lorences and Fry 1993 ). The small extent of such digestion shows that the XGO–SRs did not undergo major competing side-reactions that would have compromised our assays of XET–donor-substrate colocalization.

Known XETs, isolated from or typically expressed in expanding cells, generally have a pH optimum of ~5.5 to 6.5 (Fry et al. 1992 ; Purugganan et al. 1997 ; Campbell and Braam 1999 ; N.M. Steele and S.C. Fry, manuscript submitted). Lowering the pH of the reaction medium to 4.0 decreased the yield of xyloglucan–SR, as would be expected for a reaction catalyzed by XETs. At pH 7.0, however, the yield was not noticeably less than at pH 5.5—an observation that could point to the activity of multiple isoforms of XETs in vivo differing in pH optimum. Several XET isoforms have been described (de Silva et al. 1993 , de Silva et al. 1994 ; Okazawa et al. 1993 ; Zurek and Clouse 1994 ; Arrowsmith and de Silva 1995 ; Saab and Sachs 1995 ; Xu et al. 1995 , Xu et al. 1996 ; Steele and Fry 1999 ); in the XTR-encoding gene family of Arabidopsis, for example, the encoded proteins are 37 to 84% identical (Xu et al. 1996 ). Alternatively, the imposed high apoplastic pH could activate plasma membrane proton pumps, restoring the slightly acidic pH of the wall compartment during the assay. Finally, the pH optima of the enzymes in situ in the cell wall could also differ from those measured in vitro.

In summary, we have visualized XET–donor-substrate colocalization with excellent spatial resolution in growing cells and also in some mature tissues. The results obtained with the collenchyma, and especially with the roots, are compatible with a role for XET in elongation. This role could be wall-loosening by reversible cleavage of tethers, or it could involve the integration of new xyloglucan chains during wall assembly. These two processes probably occur concurrently in elongating cells.

With the methodology recently developed in various laboratories, it is now possible to localize the XET mRNA, the protein itself, and its action in vivo. The near future will undoubtedly bring new and important information about the regulation of cell wall assembly and loosening.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plants and Cultures
Plants of Arabidopsis thaliana (wild type) and Nicotiana tabacum cv Petite Havana SR1 were grown from seed under sterile conditions on a Murashige and Skoog medium without hormones (4.7 g/L; Duchefa, Haarlem, The Netherlands), supplemented with 10 g/L sucrose and solidified with 4 g/L Gelrite (Duchefa), pH 5.7. Arabidopsis was also grown in soil. Healthy Arabidopsis roots were obtained at various times, as explained in the text.

Celery (Apium graveolens) was obtained commercially. Celery petiole transverse sections (200 µm thick) were cut with a hand microtome (Jung, Heidelberg, Germany). The sections were then treated the same as the roots.

Cytochemical Assays
Unlabeled xyloglucan oligosaccharides (XGOs) were a mixture containing principally XLLG (nonasaccharide) > XXLG (octasaccharide) > XXXG (heptasaccharide) (see Fry et al. 1993 for nomenclature), kindly donated by K. Yamatoya (Dainippon Pharmaceutical Co., Osaka, Japan). The XGO mixture was converted to XGO–SRs (XLLG–SR > XXLG–SR > XXXG–SR, all of which act as fluorescent acceptor substrates for XETs) by the method of Fry 1997 . The XGO–SRs at 6.5 µM (sulforhodamine basis) were dissolved in 25 mM Mes buffer, pH 5.5 (different conditions are mentioned in Results). Unlabeled XGOs (~0.08 or 1 mM) were dissolved in the same buffer. Roots and celery sections were incubated in the XGO–SRs alone or supplemented with the unlabeled XGOs in the dark for 1 hr followed by a 10-min wash in ethanol/formic acid/water (15:1:4 [v/v/v]) to remove any remaining unreacted XGO–SRs; a further incubation overnight in 5% formic acid removed apoplastic, non-wall-bound xyloglucan–SR. Samples were then inspected under a fluorescence microscope (Leitz Wetzlar, Orthoplan, Germany) using green (540-nm) excitation light. Images were taken with a Nikon 35-mm camera on Fujichrome Sensia II 400 ASA film.

Chromatographic Analysis
For analysis of remaining low-M_r XGO–SRs after incubation in the presence of plant tissues for 1 hr, the plant material was rinsed, freeze-dried, and extracted with 50% ethanol; the extracts were then resolved by thin-layer chromatography (TLC) on silica gel in butan-1-ol/acetic acid/water (2:1:1[v/v/v]). The plate was illuminated with green light as above, and the fluorescent spots from the sulforhodamine conjugates were photographed through an orange filter.

	ACKNOWLEDGMENTS

K.V. is Research Assistant of the Fund for Scientific Research—Flanders (Belgium) (FWO). I.M.M.-V. was recipient of a short-fellowship from the Generalitat de Catalunya (1999BEA1200238). J.G.M. and S.C.F. were supported by a BBSRC research grant (UK).

Received February 28, 2000; accepted May 5, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Antosiewicz, D.M., Purugganan, M.M., Polisensky, D.H., and Braam, J. (1997) Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation. Plant Physiol. 115:1319-1328[Abstract].

Arrowsmith, D.A., and de Silva, J. (1995) Characterization of two tomato fruit-expressed cDNAs encoding xyloglucan endotransglycosylase. Plant Mol. Biol. 28:391-403[CrossRef][Web of Science][Medline].

Baydoun, E.A.-H., and Fry, S.C. (1989) In vivo degradation and extracellular polymer binding of xyloglucan nonasaccharide, a natural anti-auxin. J. Plant Physiol. 134:453-459.

Beemster, G.T.S., and Baskin, T.I. (1998) Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol. 116:1515-1526[Abstract/Free Full Text].

Brett, C.T., and Waldron, K.W. (1996) Physiology and Biochemistry of Plant Cell Walls. 2nd ed London, Chapman and Hall.

Campbell, P., and Braam, J. (1999) In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis. Plant J. 18:371-382[CrossRef][Web of Science][Medline].

Carpita, N.C., and Gibeaut, D.M. (1993) Structural models of primary cell walls in flowering plants: Consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3:1-30[CrossRef][Web of Science][Medline].

Catalá, C., Rose, J.K.C., and Bennett, A. (1997) Auxin regulation and spatial localization of an endo-1,4-ß-D-glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls. Plant J. 12:417-426[CrossRef][Web of Science][Medline].

Cutillas-Iturralde, A., Zarra, I., Fry, S.C., and Lorences, E.P. (1994) Implication of persimmon fruit hemicellulose metabolism in the softening process. Importance of xyloglucan endotransglycosylase. Physiol. Plant. 91:169-176[CrossRef].

de Silva, J., Jarman, C.D., Arrowsmith, D.A., Stronach, M.S., Chengappa, S., Sidebottom, C., and Reid, J.S.G. (1993) Molecular characterization of a xyloglucan-specific endo-(14)-ß-D-glucanase (xyloglucan endo-transglycosylase) from nasturtium seeds. Plant J. 3:701-711[CrossRef][Web of Science][Medline].

de Silva, J., Arrowsmith, D., Hellyer, A., Whiteman, S., and Robinson, S. (1994) Xyloglucan endotransglycosylase and plant growth. J. Exp. Bot. 45:1693-1701.

Edwards, M., Dea, I.C.M., Bulpin, P.V., and Reid, J.S.G. (1985) Xyloglucan (amyloid) mobilisation in the cotyledons of Tropaeolum majus L. seeds following germination. Planta 163:133-140[CrossRef][Web of Science].

Fanutti, C., Gidley, M.J., and Reid, J.S.G. (1993) Action of pure xyloglucan endo-transglycosylase (formerly called xyloglucan-specific endo-(14)-ß-D-glucanase) from the cotyledons of germinated nasturtium seeds. Plant J. 3:691-700[Web of Science][Medline].

Farka, V., Sulová, Z., Stratilová, E., Hanna, R., and Maclachlan, G. (1992) Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides. Arch. Biochem. Biophys. 298:365-370[CrossRef][Web of Science][Medline].

Fry, S.C. (1989a) The structure and functions of xyloglucan. J. Exp. Bot. 40:1-11[Abstract/Free Full Text].

Fry, S.C. (1989b) Cellulases, hemicellulases and auxin-stimulated growth: A possible relationship. Physiol. Plant. 75:532-536[CrossRef].

Fry, S.C. (1995) Polysaccharide-modifying enzymes in the plant cell wall. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:497-520[CrossRef][Web of Science].

Fry, S.C. (1997) Novel ‘dot-blot’ assays for glycosyltransferases and glycosylhydrolases: Optimization for xyloglucan endotransglycosylase (XET) activity. Plant J. 11:1141-1150[CrossRef][Web of Science].

Fry, S.C., Smith, R.C., Renwick, K.F., Martin, D.J., Hodge, S.K., and Matthews, K.J. (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem. J. 282:821-828.

Fry, S.C. et al. (1993) An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol. Plant. 89:1-3[CrossRef].

Hayashi, T. (1989) Xyloglucans in the primary cell wall. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:139-168[CrossRef][Web of Science].

Hayashi, T., Marsden, M.P.F., and Delmer, D.P. (1987) Pea xyloglucan and cellulose. V. Xyloglucan–cellulose interactions in vitro and in vivo. Plant Physiol. 83:384-389[Abstract/Free Full Text].

Hayashi, T., Ogawa, K., and Mitsuishi, Y. (1994a) Characterization of the adsorption of xyloglucan to cellulose. Plant Cell Physiol. 35:1199-1205[Abstract/Free Full Text].

Hayashi, T., Takeda, T., Ogawa, K., and Mitsuishi, Y. (1994b) Effects of the degree of polymerization on the binding of xyloglucans to cellulose. Plant Cell Physiol. 35:893-899[Abstract/Free Full Text].

Hetherington, P.R., and Fry, S.C. (1993) Xyloglucan endotransglycosylase activity in carrot cell suspensions during cell elongation and somatic embryogenesis. Plant Physiol. 103:987-992[Abstract].

Hoson, T., and Masuda, Y. (1991) Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins. Physiol. Plant. 82:41-47[CrossRef].

Hoson, T., Masuda, Y., Sone, Y., and Misaki, A. (1991) Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles. Plant Physiol. 96:551-557[Abstract/Free Full Text].

Iannetta, P.P.M., and Fry, S.C. (1999) Visualization of the activity of xyloglucan endotransglycosylase (XET) isoenzymes after gel electrophoresis. Phytochem. Anal. 10:238-240[CrossRef].

Koyama, T., Hayashi, T., Kato, Y., and Matsuda, K. (1983) Degradation of xyloglucan by wall-bound enzymes from soybean tissue. II. Degradation of the fragment heptasaccharide from xyloglucan and the characteristic action pattern of the -D-xylosidase in the enzyme system. Plant Cell Physiol. 24:155-162[Abstract/Free Full Text].

Labavitch, J.M., and Ray, P.M. (1974) Relationship between promotion of xyloglucan metabolism and induction of elongation by indoleacetic acid. Plant Physiol. 54:499-502[Abstract/Free Full Text].

Lorences, E.P., and Fry, S.C. (1993) Xyloglucan oligosaccharides with at least two -D-xylose residues act as acceptor substrates for xyloglucan endotransglycosylase and promote the depolymerisation of xyloglucan. Physiol. Plant. 88:105-112[CrossRef].

Maclachlan, G., and Brady, C. (1994) endo-1,4-ß-Glucanase, xyloglucanase, and xyloglucan endo-transglycosylase activities versus potential substrates in ripening tomatoes. Plant Physiol. 105:965-974[Abstract].

McQueen-Mason, S.J., Durachko, D.M., and Cosgrove, D.J. (1992) Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4:1425-1433[Abstract/Free Full Text].

McQueen-Mason, S.J., Fry, S.C., Durachko, D.M., and Cosgrove, D.J. (1993) The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls. Planta 190:327-331[Web of Science][Medline].

Nishitani, K. (1992) A novel method for detection of endo-xyloglucan transferase. Plant Cell Physiol. 33:1159-1164[Abstract/Free Full Text].

Nishitani, K. (1997) The role of endoxyloglucan transferase in the organization of plant cell walls. Int. Rev. Cytol. 173:157-206[Web of Science][Medline].

Nishitani, K., and Masuda, Y. (1982) Acid pH-induced structural changes in cell wall xyloglucans in Vigna angularis epicotyl segments. Plant Sci. Lett. 28:87-94.

Nishitani, K., and Tominaga, R. (1992) Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. J. Biol. Chem. 267:21058-21064[Abstract/Free Full Text].

Okazawa, K., Sato, Y., Nakagawa, T., Asada, K., Kato, I., Tomita, E., and Nishitani, K. (1993) Molecular cloning and DNA sequencing of endo-xyloglucan transferase, a novel class of glycosyltransferase that mediates molecular grafting between matrix polysaccharides in plant cell walls. J. Biol. Chem. 268:25364-25368[Abstract/Free Full Text].

Palmer, S.J., and Davies, W.J. (1996) An analysis of relative elemental growth rate, epidermal cell size and xyloglucan endotransglycosylase activity through the growing zone of ageing maize leaves. J. Exp. Bot. 47:339-347.

Potter, I., and Fry, S.C. (1993) Xyloglucan endotransglycosylase activity in pea internodes. Effects of applied gibberellic acid. Plant Physiol. 103:235-241[Abstract].

Potter, I., and Fry, S.C. (1994) Changes in xyloglucan endotransglycosylase (XET) activity during hormone-induced growth in lettuce and cucumber hypocotyls and spinach cell suspension cultures. J. Exp. Bot. 45:1703-1710.

Pritchard, J., Hetherington, P.R., Fry, S.C., and Tomos, A.D. (1993) Xyloglucan endotransglycosylase activity, microfibril orientation and the profiles of cell wall properties along growing regions of maize roots. J. Exp. Bot. 44:1281-1289[Abstract/Free Full Text].

Purugganan, M.M., Braam, J., and Fry, S.C. (1997) The Arabidopsis TCH4 xyloglucan endotransglycosylase: Substrate specificity, pH optimum, and cold tolerance. Plant Physiol. 115:181-190[Abstract].

Redgwell, R.J., and Fry, S.C. (1993) Xyloglucan endotransglycosylase activity increases during kiwifruit (Actinidia deliciosa) ripening. Plant Physiol. 103:1399-1406[Abstract].

Saab, I.N., and Sachs, M.M. (1995) Complete cDNA and genomic sequence encoding a flooding-responsive gene from maize (Zea mays L.) homologous to xyloglucan endotransglycosylase. Plant Physiol. 108:439-440[CrossRef][Web of Science][Medline].

Smith, R.C., and Fry, S.C. (1991) Endotransglycosylation of xyloglucans in plant cell suspension cultures. Biochem. J. 279:529-535.

Smith, R.C., Matthews, P.R., Schünmann, P.H.D., and Chandler, P.M. (1996) The regulation of leaf elongation and xyloglucan endotransglycosylase by gibberellin in ‘Himalaya’ barley (Hordeum vulgare L.). J. Exp. Bot. 47:1395-1404.

Steele, N.M., and Fry, S.C. (1999) Purification of xyloglucan endotransglycosylases (XETs): A generally applicable and simple method based on reversible formation of an enzyme–substrate complex. Biochem. J. 340:207-211.

Sulová, Z., Lednická, M., and Farka, V. (1995) A colorimetric assay for xyloglucan-endotransglycosylase from germinating seeds. Anal. Biochem. 229:80-85[CrossRef][Web of Science][Medline].

Sulová, Z., Takáová, M., Steele, N.M., Fry, S.C., and andFarka, V. (1998) Xyloglucan endotransglycosylase: Evidence for the existence of a relatively stable glycosyl–enzyme intermediate. Biochem. J. 330:1475-1480.

Thompson, J.E., and Fry, S.C. (2000) Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells. Planta in press.

Thompson, J.E., Smith, R.C., and Fry, S.C. (1998) Xyloglucan undergoes inter-polymeric transglycosylation during binding to the plant cell wall in vivo: Evidence from ¹³C/³H dual labelling and isopycnic centrifugation in caesium trifluoroacetate. Biochem. J. 327:699-708.

Valent, B.S., and Albersheim, P. (1974) The structure of plant cell walls. V. On the binding of xyloglucan to cellulose fibers. Plant Physiol. 54:105-108[Abstract/Free Full Text].

Wu, Y., Spollen, W.G., Sharp, R.E., Hetherington, P.R., and Fry, S.C. (1994) Root growth maintenance at low water potentials. Plant Physiol. 106:607-615[Abstract].

Xu, W., Purugganan, M.M., Polisensky, D.H., Antosiewicz, D.M., Fry, S.C., and Braam, J. (1995) Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase. Plant Cell 7:1555-1567[Abstract].

Xu, W., Campbell, P., Vargheese, A.K., and Braam, J. (1996) The Arabidopsis XET-related gene family: Environmental and hormonal regulation of expression. Plant J. 9:879-889[CrossRef][Web of Science][Medline].

Zurek, D.M., and Clouse, S.D. (1994) Molecular cloning and characterization of a brassinosteroid-regulated gene from elongating soybean (Glycine max L.) epicotyls. Plant Physiol. 104:161-170[Abstract].

This article has been cited by other articles:

				E. J. Mellerowicz, P. Immerzeel, and T. Hayashi Xyloglucan: The Molecular Muscle of Trees Ann. Bot., November 1, 2008; 102(5): 659 - 665. [Abstract] [Full Text] [PDF]

				V. S. T. Van Sandt, D. Suslov, J.-P. Verbelen, and K. Vissenberg Xyloglucan Endotransglucosylase Activity Loosens a Plant Cell Wall Ann. Bot., December 1, 2007; 100(7): 1467 - 1473. [Abstract] [Full Text] [PDF]

				N. Nishikubo, T. Awano, A. Banasiak, V. Bourquin, F. Ibatullin, R. Funada, H. Brumer, T. T. Teeri, T. Hayashi, B. Sundberg, et al. Xyloglucan Endo-transglycosylase (XET) Functions in Gelatinous Layers of Tension Wood Fibers in Poplar--A Glimpse into the Mechanism of the Balancing Act of Trees Plant Cell Physiol., June 1, 2007; 48(6): 843 - 855. [Abstract] [Full Text] [PDF]

				V. S. T. Van Sandt, H. Stieperaere, Y. Guisez, J.-P. Verbelen, and K. Vissenberg XET Activity is Found Near Sites of Growth and Cell Elongation in Bryophytes and Some Green Algae: New Insights into the Evolution of Primary Cell Wall Elongation Ann. Bot., January 1, 2007; 99(1): 39 - 51. [Abstract] [Full Text] [PDF]

				T. Jimenez, I. Martin, E. Labrador, and B. Dopico The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells J. Exp. Bot., December 1, 2006; 57(15): 3979 - 3988. [Abstract] [Full Text] [PDF]

				V. S. T. Van Sandt, Y. Guisez, J.-P. Verbelen, and K. Vissenberg Analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) from the lycopodiophyte Selaginella kraussiana suggests that XTH sequence characteristics and function are highly conserved during the evolution of vascular plants J. Exp. Bot., September 1, 2006; 57(12): 2909 - 2922. [Abstract] [Full Text] [PDF]

				A. WALTER and U. SCHURR Dynamics of Leaf and Root Growth: Endogenous Control versus Environmental Impact Ann. Bot., May 1, 2005; 95(6): 891 - 900. [Abstract] [Full Text] [PDF]

				K. Vissenberg, S. C. Fry, M. Pauly, H. Hofte, and J.-P. Verbelen XTH acts at the microfibril-matrix interface during cell elongation J. Exp. Bot., February 1, 2005; 56(412): 673 - 683. [Abstract] [Full Text] [PDF]

				K. Vissenberg, M. Oyama, Y. Osato, R. Yokoyama, J.-P. Verbelen, and K. Nishitani Differential Expression of AtXTH17, AtXTH18, AtXTH19 and AtXTH20 Genes in Arabidopsis Roots. Physiological Roles in Specification in Cell Wall Construction Plant Cell Physiol., January 15, 2005; 46(1): 192 - 200. [Abstract] [Full Text] [PDF]

				K. Matsui, K. Hiratsu, T. Koyama, H. Tanaka, and M. Ohme-Takagi A Chimeric AtMYB23 Repressor Induces Hairy Roots, Elongation of Leaves and Stems, and Inhibition of the Deposition of Mucilage on Seed Coats in Arabidopsis Plant Cell Physiol., January 15, 2005; 46(1): 147 - 155. [Abstract] [Full Text] [PDF]

				A. Jan, G. Yang, H. Nakamura, H. Ichikawa, H. Kitano, M. Matsuoka, H. Matsumoto, and S. Komatsu Characterization of a Xyloglucan Endotransglucosylase Gene That Is Up-Regulated by Gibberellin in Rice Plant Physiology, November 1, 2004; 136(3): 3670 - 3681. [Abstract] [Full Text] [PDF]

				E. Ramirez-Parra, M. A. Lopez-Matas, C. Frundt, and C. Gutierrez Role of an Atypical E2F Transcription Factor in the Control of Arabidopsis Cell Growth and Differentiation PLANT CELL, September 1, 2004; 16(9): 2350 - 2363. [Abstract] [Full Text] [PDF]

				Y. Guan and E. A. Nothnagel Binding of Arabinogalactan Proteins by Yariv Phenylglycoside Triggers Wound-Like Responses in Arabidopsis Cell Cultures Plant Physiology, July 1, 2004; 135(3): 1346 - 1366. [Abstract] [Full Text] [PDF]

				P. Johansson, H. Brumer III, M. J. Baumann, A. M. Kallas, H. Henriksson, S. E. Denman, T. T. Teeri, and T. A. Jones Crystal Structures of a Poplar Xyloglucan Endotransglycosylase Reveal Details of Transglycosylation Acceptor Binding PLANT CELL, April 1, 2004; 16(4): 874 - 886. [Abstract] [Full Text] [PDF]

				R. Yokoyama, J. K.C. Rose, and K. Nishitani A Surprising Diversity and Abundance of Xyloglucan Endotransglucosylase/Hydrolases in Rice. Classification and Expression Analysis Plant Physiology, March 1, 2004; 134(3): 1088 - 1099. [Abstract] [Full Text] [PDF]

				S.-J. Ji, Y.-C. Lu, J.-X. Feng, G. Wei, J. Li, Y.-H. Shi, Q. Fu, D. Liu, J.-C. Luo, and Y.-X. Zhu Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array Nucleic Acids Res., May 15, 2003; 31(10): 2534 - 2543. [Abstract] [Full Text] [PDF]

				D.-K. Lee, J. H. Ahn, S.-K. Song, Y. D. Choi, and J. S. Lee Expression of an Expansin Gene Is Correlated with Root Elongation in Soybean Plant Physiology, March 1, 2003; 131(3): 985 - 997. [Abstract] [Full Text] [PDF]

				W. H. VRIEZEN, Z. ZHOU, and D. VAN DER STRAETEN Regulation of Submergence-induced Enhanced Shoot Elongation in Oryza sativa L. Ann. Bot., January 2, 2003; 91(2): 263 - 270. [Abstract] [Full Text] [PDF]

				K. Vissenberg, V. Van Sandt, S. C. Fry, and J-P. Verbelen Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays J. Exp. Bot., January 2, 2003; 54(381): 335 - 344. [Abstract] [Full Text] [PDF]

				V. Demidchik, S. N. Shabala, K. B. Coutts, M. A. Tester, and J. M. Davies Free oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in plant root cells J. Cell Sci., January 1, 2003; 116(1): 81 - 88. [Abstract] [Full Text] [PDF]

				J. K. C. Rose, J. Braam, S. C. Fry, and K. Nishitani The XTH Family of Enzymes Involved in Xyloglucan Endotransglucosylation and Endohydrolysis: Current Perspectives and a New Unifying Nomenclature Plant Cell Physiol., December 15, 2002; 43(12): 1421 - 1435. [Abstract] [Full Text] [PDF]

				V. Bourquin, N. Nishikubo, H. Abe, H. Brumer, S. Denman, M. Eklund, M. Christiernin, T. T. Teeri, B. Sundberg, and E. J. Mellerowicz Xyloglucan Endotransglycosylases Have a Function during the Formation of Secondary Cell Walls of Vascular Tissues PLANT CELL, December 1, 2002; 14(12): 3073 - 3088. [Abstract] [Full Text] [PDF]

				K. Vissenberg, S. C. Fry, and J.-P. Verbelen Root Hair Initiation Is Coupled to a Highly Localized Increase of Xyloglucan Endotransglycosylase Action in Arabidopsis Roots Plant Physiology, November 1, 2001; 127(3): 1125 - 1135. [Abstract] [Full Text] [PDF]

				R. Yokoyama and K. Nishitani A Comprehensive Expression Analysis of all Members of a Gene Family Encoding Cell-Wall Enzymes Allowed us to Predict cis-Regulatory Regions Involved in Cell-Wall Construction in Specific Organs of Arabidopsis Plant Cell Physiol., October 1, 2001; 42(10): 1025 - 1033. [Abstract] [Full Text] [PDF]

				T. A. Wagner and B. D. Kohorn Wall-Associated Kinases Are Expressed throughout Plant Development and Are Required for Cell Expansion PLANT CELL, February 1, 2001; 13(2): 303 - 318. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via ISI Web of Science (66) Citing Articles via Google Scholar Google Scholar Articles by Vissenberg, K. Articles by Fry, S. C. Search for Related Content PubMed PubMed Citation Articles by Vissenberg, K. Articles by Fry, S. C. Agricola Articles by Vissenberg, K. Articles by Fry, S. C.