Relationship between Promotion of Xyloglucan Metabolism and Induction of Elongation by Indoleacetic Acid

Abstract

Auxin promotes the liberation of a xlyoglucan polymer from the cell walls of elongating pea (Pisum sativum) stem segments. The released polymer can be isolated from the polysaccharide fraction of the water-soluble portion of tissue homogenates, thus providing as assay for this kind of metabolism. Promotion of xyloglucan metabolism by auxin begins within 15 minutes of hormone presentation. The MATERIALS AND METHODSThe experiments followed the pulse-chase protocol previously described in detail (9). Briefly, samples of segments 8 mm long, cut from the stems of 7-day-old etiolated pea seedlings (Pisum sativum, var. Alaska), were depleted of endogenous substrates during a 1-hr incubation in water, labeled during a 1-hr incubation in uniformly labeled "C-glucose, and then treated for 3 hr in 50 mm unlabeled glucose to dilute radioactive metabolites from internal substrate pools. Media containing 50 mm unlabeled glucose (in water), IAA (1.7 X 10-' M) and/or other agents were then supplied for an experimental treatment period, the nature and duration of which varied from experiment to experiment as described below.At the termination of treatment the samples were rinsed and chilled over ice and ground in ice water. Cell wall material was collected and washed twice by centrifugation at 1000g. Protein was precipitated from the combined supernatants using 5% trichloroacetic acid; the remaining soluble material was concentrated by lyophilization and made 80% in ethanol at 2 C to precipitate water-soluble polysaccharides. These were redissolved in water and passed through Dowex-1-acetate. Neutral components were washed through with water, then acidic components were eluted with 6 N acetic acid. These effluents were concentrated by lyophilization and their radioactivity was determined by scintillation spectrometry. Radioactivity in the neutral subfraction is reported as watersoluble xyloglucan.Unless otherwise noted, radioactivity values for each time point or each treatment or both were determined from triplicate samples of 30 pea-stem segments (initial fresh weight, 510 mg). Hydrolysis and chromatography for determining composition of fractions followed methods described previously (8,9). RESULTSWhen the cell walls of pea stem tissue are labeled by feeding "C-glucose, and the tissue is then given IAA under "chase" conditions that prevent appreciable further incorporation of "C from soluble pools, IAA causes a substantial increase in "C-xylose and "C-glucose of water-soluble polysaccharides (9). This increase was inferred to represent a liberation of polymer(s) from the cell wall. Table I shows that when this watersoluble polysaccharide material is separated into neutral and acidic components its "C-glucose and "4C-xylose appear almost exclusively in the neutral subfraction, which contains, in addition, a small amount of "4C-galactose. The IAA effect likewise appears entirely in the neutral subfraction.Determination of radioactivity in the neutral water-soluble polysaccharide subfraction affords an assay for the I...

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate

“…The enzymic restructuring of cell-wall polysaccharides in i o is widely held to be important in the control of plant growth [1][2][3], abscission [4] and fruit ripening [5][6][7]. Wall enzymes that could restructure polysaccharides include hydrolases and transglycosylases [3].…”

“…However, the mere presence of these enzymes in a cell wall does not prove that they catalyse reactions there [3]. To test whether the enzymes do act in the walls of living cells, it is necessary to monitor the metabolism of their polysaccharide substrates in i o. Hydrolase action would decrease the M r of wall polysaccharides, and such decreases have indeed been seen during normal growth and in response to exogenous auxins or low pH [1,2,8]. However, there is currently no evidence that wall polysaccharides undergo transglycosylation in i o.…”

“…Abbreviations used : CsTFA4 (Pharmacia), caesium trifluoroacetate ; GPC, gel-permeation chromatography ; PyAW, pyridine/acetic acid/water (1 : 1 : 23, by vol., pH 4.7) ; ∆ρ, difference in buoyant density ; note that, in the names of isotopically labelled compounds, 1 H and 12 C are omitted except for emphasis ; (c)XET, (cauliflower) xyloglucan endotransglycosylase ; XLLGol, the structure of this oligosaccharide is given in the text. 1 Present address : Department of Crop Science and Technology, Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, U.K. 2 To whom correspondence should be addressed.…”

“…1 Present address : Department of Crop Science and Technology, Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, U.K. 2 To whom correspondence should be addressed. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET.…”

Viscosimetric determination of cellulase activity: critical analyses

“…This indicates that the results obtained with CMC or HEC must not only be necessarily related to the physiological concept of cellulose, but also to hemicellulose, etc. Moreover, xyloglucan -perhaps a "key" compound of plant cell walls, when explaining cell extension -has a chemical structure similar to that of HEC (12,14) and it can be hydrolysed by a cellulase mixture (13,23). Consequently, when using HEC to test cellulase activity, it would be difficult to conclude simply that this system does not possibly include a xyloglucanase which seems to be an essential enzyme for controlling growth.…”