lan Potter and Stephen C. Fry*. Daniel Rutherford Building, University of Edinburgh, The King's Buildings, Mayfield Road,. Edinburgh EH9 3JH, United Kingdom.
Plant Physiol. (1 993) 103: 235-241
Xyloglucan Endotransglycosylase Activity in Pea Interndes’ Effects of Applied Gibberellic Acid lan Potter and Stephen C. Fry*
Daniel Rutherford Building, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JH, United Kingdom
to the mechanism of auxin-induced wall loosening (Labavitch and Ray, 1974; Hoson et al., 1991). Xyloglucan is a wallbound hemicellulose with a backbone of /?-(1+ 4)-linked DGlc residues, about 60 to 75% of which are a-D-xylosylated at 0-6; galactosyl, fucosyl, and O-acetyl groups are also present (for review, see Fry, 1989a). Xyloglucan chains, which may be about 1 pm in total length, can hydrogen-bond to cellulose (Hayashi et al., 1987) and may act as molecular tethers between adjacent cellulosic microfibrils in the cell wall, restraining wall expansion (Fry, 1989b; Passioura and Fry, 1992). The evidence that xyloglucan participates in auxin-induced wall loosening is 2-fold. First, during auxininduced growth, xyloglucan decreases in mean M, and may become partially solubilized, suggesting endohydrolysis catalyzed by cellulase (EC 3.2.1.4) (Labavitch and Ray, 1974; Wakabayashi et al., 1991). Second, treatment of dicotyledonous stem segments with xyloglucan-binding lectins or antibodies, which might be expected to interfere with xyloglucanenzyme interaction, can block auxin-induced growth (Hoson et al., 1991). If xyloglucan cleavage occurred solely by cellulase-catalyzed hydrolysis, the repeated cutting of tethers might be expected to reduce wall strength unduly; therefore, a repair function would seem equally important. Endotransglycosylation could provide such a mechanism, and an enzyme, XET, capable of catalyzing xyloglucan endotransglycosylation, has been described recently (McDougall and Fry, 1990; Smith and Fry, 1991; Farka; et al., 1992; Fry et al., 1992b; Nishitani and Tominaga, 1992). XET cuts a xyloglucan molecule (the donor substrate) in mid-chain and then conserves the energy of the cleaved glucosyl bond in the formation of a new, chemically identical bond with another xyloglucan or XGO molecule (the acceptor substrate). The reaction can be monitored by measurement of the incorporation of radioactively labeled XGOs (Smith and Fry, 1991) or fluorescently labeled XGOs (Nishitani and Tominaga, 1992) into high-Mr xyloglucan, or by determination of changes in the size distribution of high-Mr xyloglucan molecules (McDougall and Fry, 1990;
Xyloglucan endotransglycosylase (XET) activity extractable from internodes of tal1 and dwarf varieties of pea (Pisum sativum 1.) was assayed radiochemically using tamarind seed xyloglucan as donor substrate and an olig~saccharidyl-[~H]alditol as acceptor substrate. lnternodes I and II showed little elongation during the period 15 to 21 d after sowing; XET activity remained relatively constant and was unaffected by exogenous gibberellic acid (CA,). A single application of CA3 to the dwarf genotype resulted in a small enhancement of elongation in internode 111 between d 17 and 21 and caused a small increase in XET activity in internode 111. Repeated applications of CA3 caused internode V to elongate between d 20 and 26, to the same extent as in the tal1 variety, and concomitantly led to greatly elevated XET activity (expressed per unit fresh weight, per unit of extractable protein, and per internode). Thus, XET activity correlated with CA3-enhanced length in pea internodes; the possibility that this represents a causal relationship is discussed.
GAs have been shown to regulate intemode elongation in many plants, especially in dwarf varieties (Jones, 1973; Ross et al., 1992). Internode elongation is due to turgor-driven wall yielding (Cleland, 1981; Cosgrove, 1986). Therefore, GAs could enhance elongation by increasing either wall extensibility or turgor pressure. Several studies show that GAs increase wall extensibility (Nakamura et al., 1975; Stuart and Jones, 1977; Cosgrove and Sovonick-Dunford, 1989). Several biochemical mechanisms have been proposed for the effects of GAs on wall extensibility,including a promotion of wall-loosening reactions (Cleland, 1981; Cosgrove and Sovonick-Dunford, 1989), an inhibition of wall-tightening reactions (Fry, 1980), changes in the relative rates of synthesis of specific wall polysaccharides (e.g. a relative increase in cellulose synthesis [Mondal, 1975]), and increased total wall synthesis (Montague and Ikuma, 1975). The rapidity with which GAs can initiate growth promotion (within 10 min in lettuce hypocotyls [Moll and Jones, 19811) focuses attention on wall-loosening reactions, at least in studies of the early phases of the response. Studies of GA-induced wall loosening may reasonably draw inspiration from studies of auxin action. In dicotyledonous plants, the metabolism of xyloglucan appears to be central
Abbreviations: XET, xyloglucan endotransglycosylase; XGO, xyloglucan-derived oligosaccharide; XXXGol, a-o-xylopyranosyl-(1 --* 6)-P-o-ghcopyranosy1-(1+ 4)-[a-~-xy~opyranosy~-( 1+ 6)]-P-o-glu+ 6)]-P-~-glucopyranocopyranosyl-(1 + 4)-[a-~-xylopyranosyl-(l syl-(1 + 4)-o-glucitol.
‘ Supported by the Agricultura1and Food Research Council * Corresponding author; fax 44-31-650-5392. 235
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Potter and Fry
Farkai et al., 1992; Nishitani and Tominaga, 1992; Lorences and Fry, 1993). Severa1 correlations exist between XET activity and tissue growth rate, e.g. during the growth cycle of spinach cell cultures and along the length of an elongating stem or root (Fry et al., 1992a; Pritchard et al., 1993). However, no increase in XET activity occurred during the auxin-induced elongation of pea stem segments (Fry et al., 1992a, 1992b). Also, the stems of dark-grown (rapidly elongating) pea seedlings did not contain more XET activity than those of lightgrown (slowly elongating) seedlings (I. Potter and S.C. Fry, unpublished observations). Furthermore, a wall-bound protein that appears to mediate H+-induced wall creep was shown to lack XET activity (McQueen-Mason et al., 1993). In the present work, we have investigated whether XET activity is correlated with GA3-induced growth. This makes an interesting comparison with the studies mentioned above because, unlike auxin, GAs do not appear to act via H+ secretion (Stuart and Jones, 1978), and endogenous GAs do not seem to be responsible for dark-induced elongation of stems (Sponsel, 1986). MATERIALS A N D METHODS Plant Material
Pea (Pisum safivum L., var Feltham First [GA-responsive dwarf] and Pilot [tall]) seeds were soaked for 24 h in running tap water before being sown in soil at 80 seeds per tray and grown for 14 d in the glasshouse. lnternodes I to 111
Fourteen days after sowing, 80 plants were sprayed with 400 to 500 mL of 0.1 m GA3 in 0.1% (v/v) DMSO or with 400 to 500 mL of 0.1% DMSO. Five plants of each treatment were randomly harvested at 24-h intervals thereafter for the following week. Successive internodes (numbered from the cotyledonary node) were measured (+1 mm), excised, and weighed. Lengths are reported as mean (+SE) of the five replicate plants. lnternode V
Since at 14 d after sowing the plants had three to four visible internodes, number V was selected for study as a posttreatment internode. After 14 d, 80 plants were sprayed as above and then resprayed every 48 h throughout the experiment. Also at 48-h intervals, five plants were randomly harvested and internode V was measured (k1mm), excised, and weighed. Extraction of Soluble XET Activity
Excised internodes were frozen (-18OC) in buffer (50 m~ succinic acid, 10 mM L-ascorbic acid, 10 m~ CaC12,and 1 m~ DTT, final pH adjusted to 5.5 with NaOH) at 2 mL/g fresh weight, thawed, and homogenized at O to 4OC by mortar and pestle with a little acid-washed sand. Homogenates were centrifuged for 10 min at 2500g and O to 4OC, and the supematant was immediately assayed.
Plant Physiol. Vol. 103, 1993
Substrates
Tamarind seed xyloglucan (high-M, donor substrate) was kindly donated by Dr. J.S.G. Reid (University of Stirling, UK). [1-3H]XXXGol (low-M, acceptor substrate; specific activity 22.5 MBq/pmol) was synthesized and kindly donated by Dr. P.R. Hetherington (Universityof Edinburgh, UK). Enzyme Assay
Substrate solution (2 mg/mL of tamarind seed xyloglucan and 75 kBq/mL of [3H]XXXGolin the above buffer; 120 pL) was mixed with 10 pL of enzyme extract and incubaied for 1 h at 25OC. The reaction was stopped by the addition of 100 pL of 20% (w/v) formic acid. The products were dried on a 5 X 5-cm square of Whatman 3MM chromatography paper, which was then washed for 45 min in running tap water to remove the unreacted [3H]XXXGol, and redried at 6OoC. The squares were placed, with the loaded side outermost, in 22mL scintillation vials, soaked with 2 mL of scintillant A [0.5% 2,5-diphenyloxazoleand 0.05% 1,4-bis(5-phenyl-2-oxazolylbenzene in toluene], and assayed for paper-bound [3H]xyloglucan by scintillation counting (efficiency was approxirnately 44%). Enzyme activity is recorded as cpm of paper-bound radioactivity produced (mg fresh weight)-' h-'. Data are plotted as the mean (+-SE) of three replicate assays Iof the combined homogenate of five internodes. Detailed data are provided for two experiments; comparable results haci been obtained in two independent previous trials (data not shown). Protein Assay
Protein was assayed by the Bio-Rad protein assay dye reagent (Coomassie blue) as recommended by the sup pliers. BSA was used as standard. Gel Permeation Chromatography
To test for possible degradation of the [3H]XXXGol,e.g. by the action of contaminating a-D-xylosidase and P-D-gblCOSidase, during the XET assays, we examined the M, rarge of the reaction products. For this work, the enzyme extract was concentrated by precipitation with saturated (NH4)2S04so that the reactions would approach completion during the 1h incubation period. Samples of the reaction products formed by concentrated extracts of internode I11 of GA3-treatetl and untreated tall and dwarf genotypes were applied to a 1.5 X 150-cm column of Bio-Gel P-2 and equilibrated and eluted with pyridine:acetic acid:water (1:1:23 by volume, pII approximately 4.5). Interna1 nonradioactive markers of dextran (M, approximately 9000), maltoheptaose, and Glc were added to the samples and detected in the eluate with anthrone. Fractions were assayed for radioactivity in 10 volumes of scintillant B [0.33% 2,5-diphenyloxazole and 0.033% 1,4bis(5-phenyl-2-oxazolyl)benzene in to1uene:Triton I(-100 ( 2 1 , v/v)]. RESULTS Validation of Assay
XET activity was found in a11 internodes tested (I, I[, 111, and V). Product formation was usually linear with respcxt to
CA3 and Xyloglucan Endotransglycosylase
time over the 1st h. Where this was not the case (in the most active extracts), the enzyme sample was diluted prior to assay, and the rate then became linear. When the rate was linear, the amount of product formed within 1 h was proportional to enzyme concentration (data not shown). Untreated [3H]XXXGol (acceptor substrate) contained no high-M, radioactive material (Fig. la). An (NH4)2S04-concentrated extract of internode 111 of the tall genotype caused the majority of the [3H]XXXGolto be incorporated into high-Mr material within 1 h (Fig. lb). Negligible degradation of the [3H]XXXGoloccurred during this reaction, as shown by the lack of radioactive material eluting later than the [3H]XXXGol (Fig. lb). Similar results were obtained for extracts of the GA3-treated and -untreated dwarf genotype (data not shown). The lack of decrease in mo1 wt of the substrate ([3H]XXXGol)allows us to discount the possibility that contaminating hydrolases interfered during the XET assay. This eliminates, for example, the possibility that cY-D-xylosidase
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Elongation and XET activity in internode I. Effect of a single application of GAs on the elongation (a) and XET activity (b) of internode I of pea. Time zero on the graphs represents the time of spraying with CA, (O,W) or with the control solution (O, O) 14 d after sowing. The plants were cv Feltham First (dwarf genotype; O, O) or cv Pilot (tal1 genotype; O, m). activity interfered during the assay by removing the essential xylose residue (Lorencesand Fry, 1993) from the nonreducing terminus of the [3H]XXXGol. Also, viscometric assays with either xyloglucan or carboxymethylcellulose as substrate showed that very little cellulase activity was present in the enzyme extracts used (data not shown).
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Figure 2.
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fraction number Figure 1. The acceptor substrate is not degraded during the XET assay. Gel-permeation chromatography, on Bio-Gel P-2, of [3H]XXXGol (a) and the reaction products (b) obtained after incubation enzyme of ['HIXXXGol for 1 h with an (NH4)2S04-concentrated extract from internode I I I of tal1 pea plants harvested 20 d after
sowing. Arrows indicate the peak positions of interna1 nonradioactive markers; M7, maltoheptaose.
Intemode I showed very slight GA3-induced elongation in the dwarf genotype (Fig. 2a). Internode I of the tall genotype was consistently 2 cm longer than in the dwarf. Extractable XET activity showed little variation with time, either between genotypes or in response to GA3. This was true whether the data were expressed as activity per unit fresh weight (Fig. 2b) or as specific activity (not shown). Internode 11
Intemode I1 also showed little elongation, except in the untreated tall genotype, where elongation continued until96
Potter and Fry
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h after spraying (Fig. 3a). Internode I1 of the tall genotype was 2 to 3 cm longer than in the dwarf. At 24 h after spraying, extractable XET activity (per unit fresh weight) was higher in the tall than in the dwarf genotype, but by 96 h, the difference had become indiscernible (Fig. 3b). At 96 to 168 h postspraying, the XET activity per unit fresh weight of internode I1 was about one-third higher than in intemode I.
Plant Physiol. Vol. 103, 1993
10
This was the major elongating intemode in the first few days after spraying (Fig. 4a). In the tall genotype, internode I11 elongated throughout the period of observation; the rate of elongation was unaffected by GA3. The untreated dwarf showed much less elongation. GA3 treatment of the dwarf genotype moderately increased the elongation of intemode 111. Extractable XET per unit fresh weight (Fig. 4b) and per mg extractable protein (data not shown) increased in a11 treatments in parallel with growth. Genetically tall plants had consistently higher XET activities than the untreated dwarf; GA3-treateddwarf plants had an intermediate activity. XET activity per unit fresh weight in intemode I11 was initially much lower than in intemode 11, but the activities in the two intemodes tended to equalize as intemode I11 grew.
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time (hl Figure 4. Elongation and XET activity in internode I I I . Effect of a single application of CA3 on t h e elongation (a) and XET activity (b) of internode I I I of pea. Other details are t h e same as for Figure 2.
Internode V
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Internode V did not appear until a few days after spraying. Its growth response was similar to that of internode 111. but GA3 caused internode V of the dwarf genotype to reach a final length equal to that of the tall genotype (Fig. 5a). Length was closely correlated with XET activity per unit fresh weight (Fig. 5b); the GA3-treated and -untreated tall genotype and the GA3-treated dwarf genotype showed broadly similar XET values at each sampling point. The untreated dwarf, which showed little elongation, had a consistently low XET activity per unit fresh weight (Fig. 5b). The specific activity of XET extractable from intemode V also correlated with length (Fig. 5c), indicating a selective enhancement of the accumulation of active XET relative to other extractable proteins. Because internode V had a substantially greater Sresh weight in the tall and GA3-treated dwarf genotypes than in the untreated dwarf, it follows from the data in Figure 5b that the XET activity per internode was very much higher in the tall and GA3-treated dwarf plants (Fig. 5d). At 240 h, the GA3-treated dwarf genotype had about 80-fold more XET activity per intemode V than did the untreated dwarf.
2 1
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time (hl Figure 3. Elongation and XET activity in internode II. Effect of a single application of GA, on the elongation (a) and XET activity (b) of internode II of pea. Other details are t h e same as for Figure 2.
DISCUSSION To our knowledge, the present work reports the largest known GA-induced effect on the activity of a poteniially
GA3 and Xyloglucan Endotransglycosylase
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time (hl Elongation and XET activity in internode V. Effect of multiple applications of GA, on internode V of pea. a, Elongation; b, XET activity per unit fresh weight; c, XET specific activity; d, XET activity per internode. Other details are the same as for Figure 2.
Figure 5 .
wall-loosening enzyme. We have demonstrated a strong positive correlation between GA3-enhanced length and extractable XET activity in pea intemodes. XET activity and specific activity increased 3- to 6-fold during elongation (whether endogenous in the tal1 genotype or GA3-induced in the dwarf), while remaining at a constant, lower leve1 in internodes that had stopped growing. Any potentially wall-loosening enzyme could conceivably mediate the action of GA3. Cellulase (EC 3.2.1.4), which hydrolyzes xyloglucan more readily than native cellulose (Hayashi et al., 1984), was induced in GA3-treated abscission zones (Rainer et al., 1969) and in auxin-treated pea stems (Fan and Maclachlan, 1967). However, in dwarf pea internodes, GA3 provoked a 30% decrease in cellulase specific 4 000000000000000000000000001)
+
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activity (Broughton and McComb, 1971). ln abscission zones, GA3 appeared to promote the hydrolysis of pectic polysaccharides (Bomman et al., 1969); this has not been reported in growing tissues, although a 25% reduction in pectinmethylesterase specific activity was noted in GA3-treated dwarf pea internodes (Broughton and McComb, 1971). Thus, our data suggest that XET activity may be particularly relevant to an understanding of GA action. Further work will be required to determine whether the increased XET activity is causally involved in the enhancement of elongation during the non-H+-mediated action of GA3.It will also be important to determine whether the increase in XET activity is due to an increase in the synthesis of the XET protein or to an inhibition of its degradation or denaturation. GA3 treatment
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Potter and Fry
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of dwarf pea stems does promote the de novo synthesis of several unidentified soluble proteins (Chory et al., 1987); however, none of these had M, close to 33,000, the size reported for azuki bean XET (Nishitani and Tominaga, 1992). It would be oversimplistic to propose that XET is the sole determinant of wall loosening during GA3-induced growth; in particular, the action of other enzymes on wall-bound xyloglucans may also contribute. For example, cellulase can hydrolyze xyloglucan and this could loosen the wall. Also, the products of cellulase action include XGOs, the formation of which has been detected in vivo (McDougall and Fry, 1991). These XGOs are good acceptor substrates for transglycosylation in a reaction in which XET effectively becomes a xyloglucan-cleaving enzyme’ (Scheme 1) rather than an enzyme involved in the cleavage and subsequent repair of xyloglucan chains, as occurs when polysaccharide is the acceptor (Scheme 2). This means that cellulase, as well as directly cutting xyloglucan chains by endohydrolysis, can also convert XET into a “chain-breaking” enzyme by supplying it with low-M, acceptor molecules (XGOs). The scission of xyloglucan chains, either by XET in the presence of cellulase-generated XGOs or by cellulase directly, could loosen the cell wall in a manner epistatic to the cleave-and-repair action that XET exerts when it uses a polysaccharide as acceptor substrate. Other enzymes that could modify the action of XET in vivo include a-D-xylosidase fKoyama et al., 1983), which, by removing a single xylose group from the nonreducing end of a xyloglucan molecule (Eq. 1,reaction a), could greatly diminish its ability to act as an acceptor substrate for XET (Lorences and Fry, 1993). Subsequent action of P-D-glucosidase (Eq. 1, reaction b) could restore acceptor function by regenerating the necessary acceptor structure:
Xyl
L
Xyl
L
Xyl
(a)
L
G 1c+G 1c+G 1c+Glc+.
-+
..
Plant Physiol. Vol. 103, 1993
tions of XET, cellulases, a-D-xylosidase, and P-D-glucos*idase, which act to modify the structure of xyloglucan, procluce a complicated picture of xyloglucan metabolism. The iniricate relationship that exists between these enzymes and other wall enzymes such as peroxidase could have a procound influence on wall loosening and, thus, on growth. Despite this great potential intricacy, however, we believe that the results reported in the present paper invite attention to be focused on XET in future attempts to explain the bajsis of GA3-promoted growth. Received March 8, 1993; accepted May 30, 1993. Copyright Clearance Center: 0032-0889/93/103/0235/07 LITERATURE ClTED
Bornman CH, Spurr AR, Addicott FT (1969) Histochemica:l localization by electron microscopy in abscising tissue. J S Afr Bot 3 5 253-264 Broughton WJ, McComb AJ (1971) Changes in the pattern of enzyme development in gibberellin-treated pea internode:j. Ann Bot 3 5 213-228 Chory J, Voytas DF, Olszewski NE, Ausubel FM (1987) Gibberellin-induced changes in the populations of translatable mRN.4s and accumulated polypeptides in dwarfs of maize and pea. Plant Physiol83: 15-23 Cleland RE (1981) Wall extensibility: hormones and wall extension. In W Tanner, FA Loewus, eds, Plant Carbohydrates 11: Extracellular Carbohydrates. Encyclopedia of Plant Physiology New Seri.es:Vol 13B. Springer, Berlin, pp 255-276 Cosgrove DJ (1986) Biophysical control of plant cell growth. Annu Rev Plant Physiol36: 377-405 Cosgrove DJ, Sovonick-Dunford SA (1991) Mechanism of 8bberellin-dependent stem elongation in peas. Plant Physiology 8 9 184-191 Fan D-F, Maclachlan GA (1967) Studies on the regulation of cellulase activity and growth in excised pea epicotyl sections. Can J Bot 4 5 1837-1844 FarkaB V, Sulova Z, Stratilova E, Hanna R, Maclachlan G (1992) Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides. Arch Biochem Biophys 298: 365-370 Fry SC (1980) Gibberellin-controlled pectinic acid and protein secretion in growing cells. Phytochemistry 1 9 735-740 Fry SC (1989a) The structure and functions of xyloglucan. J Exp Bot 40: 1-11
In addition to the complicated interplay of reactions involving xyloglucan, other apparently unrelated reactions could contribute to GA3-inducedwall loosening. For example, several GAs decrease the secretion of peroxidase into the cell wall (Fry, 1980) and could thereby minimize the formation of oxidatively coupled phenolic cross-links (eg., diferulate and isodityrosine). Thus, a decrease in peroxidase could delay the tightening of the cell wall, and an increase in XET could promote its loosening. The wall-tightening action of peroxidase could perhaps explain why growth eventually ceases in older intemodes even though XET activities remain high. The combined ac-
* In these diagrams, each circle or square denotes one XGO (e.g. heptasaccharide) unit; circles, donor chain; squares, acceptor; filled symbols, reducing terminal XGO unit; J, bond cleaved.
Fry SC (1989b) Cellulases, hemicelluloses and auxin-stimulated growth: a possible relationship. Physiol Plant 7 5 532-536 Fry SC, Smith RC, Hetherington PR, Potter I (1992a) Endotransglycosylation of xyloglucan: a role in cell wall yielding? Curr Top Plant Biochem Physiol 11: 42-62 Fry SC, Smith RC, Renwick KF, Martin DJ, Hodge SK, Matthews KG (1992b) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem J 282: 821-828 Hayashi T, Marsden MPF, Delmer DP (1987) Pea xyloglucan and cellulose. V. Xyloglucan-celluloseinteractions in vitro and iri vivo. Plant Physiol83: 384-389 Hayashi T, Wong Y, Maclachlan G (1984) Pea xyloglucan and cellulose. 11. Partia1 hydrolysis by pea endo-1,4-(3-glucanases..Plant Physiol75 605-610 Hoson T, Masuda Y, Sone Y, 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 Jones RL (1973) Gibberellins: their physiological roles. Annu Rev Plant Physiol24 571-598 Koyama T, Hayashi T, Kato Y, Matsuda K (1983) Degradation of xyloglucan by wall-bound enzymes from soybean tissue. 11. Degradation of the fragment heptasaccharide from xyloglucan and the characteristic action pattern of the a-o-xylosidase in the erizyme system. Plant Cell Physiol 2 4 155-162
GA, and Xyloglucan Endotransglycosylase Labavitch J, Ray PM (1974) Relationship between promotion of xyloglucan metabolism and induction of elongation by indoleacetic acid. Plant Physiol 5 4 499-502 Lorences EP, Fry SC (1993) Xyloglucan oligosaccharides with at least two a-D-xylose residues act as acceptor substrates for xyloglucan endotransglycosylase and promote the depolymerisation of xyloglucan. Physiol Plant 88: 105-1 12 McDougall GJ, Fry SC (1990) Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion. Plant Physiol93: 1042-1048 McDougall GJ, Fry SC (1991) Xyloglucan nonasaccharide, a naturally-occurring oligosaccharin, arises in vivo by polysaccharide breakdown. J Plant Physiol137: 332-336 McQueen-Maso S, Fry SC, Durachko DM, Cosgrove DJ (1993) The relationship between xyloglucan endotransglycosylase and in vitro cell wall extension in cucumber hypocotyls. Planta 190 327-331 Mo11 C, Jones RL (1981) Short-term kinetics of elongation growth of gibberellin-responsive lettuce hypocotyl sections. Planta 152: 442-449 Monda1 MH (1975) Effects of gibberellic acid, calcium, kinetin, and ethylene on growth and cell wall composition of pea epicotyls. Plant Physiol 56: 622-625 Montague MJ, Ikuma H (1975) Regulation of cell wall synthesis in Avena stem segments by gibberellic acid. Plant Physiol 5 5 1043-1047 Nakamura T, Sekine S, Arai K, Takahashi N (1975) Effect of gibberellic acid and indole-3-acetic acid on stress relaxation properties of pea hook cell wall. Plant Cell Physiol 1 6 127-138 Nishitani K, Tominaga R (1992) Endo-xyloglucan transferase, a
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