Auxin extractable

During seed development auxins accumulate in the nutritive tissues, i.e. in the endosperm and nucellus. This was shown firstly for the cereals (e.g. rye [78]) and later for apple [107] and garden pea [51] among others. In the garden pea the auxin produced in the endosperm was found to be 4-chloroindole acetic acid, whereas its methyl ester occurred mainly in the embryo [65]. The auxin extractable from the seed tissues is almost certainly produced there, presumably by the biosynthetic pathway which has been worked out from studies on other parts of higher plants. These have shown that lAA is derived from the amino acid tryptophan, by the route shown in Figure 3.28. Further details can be found in recent reviews (e.g. [158]). 

Abscisin II is a plant hormone which accelerates (in interaction with other factors) the abscission of young fruit of cotton. It can accelerate leaf senescence and abscission, inhibit flowering, and induce dormancy. It has no activity as an auxin or a gibberellin but counteracts the action of these hormones. Abscisin II was isolated from the acid fraction of an acetone extract by chromatographic procedures guided by an abscission bioassay. Its structure was determined from elemental analysis, mass spectrum, and infrared, ultraviolet, and nuclear magnetic resonance spectra. Comparisons of these with relevant spectra of isophorone and sorbic acid derivatives confirmed that abscisin II is 3-methyl-5-(1-hydroxy-4-oxo-2, 6, 6-trimethyl-2-cyclohexen-l-yl)-c s, trans-2, 4-pen-tadienoic acid. This carbon skeleton is shown to be unique among the known sesquiterpenes. 

S. Nardi, M. R. Panuccio, M. R. Abenavoli, and A. Muscolo, Auxin-like effect of humic substances extracted from faeces oi Allolohophora Caliginosa and A. rosea. Soil Biol. Biochem. 26 1341 (1994). 

In the belief that the failure of the stem section to grow meant that some essential cofactor must normally be supplied to the stem section by the rest of the plant, we began investigating the effect of plant extracts. A mixture of glycerides isolated from the pea nearly doubled the growth response of the section to auxin and gibberellin, even when applied at the extremely low concentration of 10 mg. per liter (3). Even so, the growth attained by the section is still less than that of the intact plant, and a cofactor other than those discussed in this paper may be involved. 

Enzyme Fractionation. Buffer-soluble and -insoluble cellulases from auxin-treated apices of pea epicotyls were extracted in a crude form and purified to homogeneity as previously described (3). For fractionation, a Sephadex G-100 column (1.6 X 100 cm) was prepared and equilibrated with 20mM sodium phosphate, pH 6.2, containing 5% glycerol and 0.03% sodium azide at 2°C. Crude enzyme preparations (1.5 mL) containing... 

Physical Properties. All of the cellulase (CMCase) activity which develops in auxin-treated pea apices dissolves in salt solutions (e.g., phosphate buffer, 20mM, pH 6.2, containing 1M NaCl). Gel chromatography of such extracts indicates the presence of two cellulase components with similar levels of activity and elution volumes corresponding to molecular weights of about 20,000 and 70,000 (Figure 1). If the tissue is extracted with buffer alone, only the smaller cellulase dissolves (referred to as buffer-soluble or BS cellulase). The larger buffer-insoluble (BI) cellulase can then be extracted from the residue by salt solutions. This simple extraction procedure effectively separates the two cellulases, and can be used as an initial step for their estimation or purification. 

Figure 1. Elution profiles of cellulase activity from Sephadex G-100 gel chromatographs of crude extracts of auxin-treated pea apices. BS cellulose activity has an elution volume corresponding to a molecular weight of 20,000. BI cellulase activity dissolves in 1M NaCl and elutes with a molecular weight of 70,000. These values correspond to those observed for purified cellulases (3), indicating that the enzymes were not altered in molecular weight during purification, and could be effectively separated by differential extraction.

Figure 3. Development of BS and BI cellulase activity in apices of pea seedlings. Intact seedlings were sprayed with the auxin analogue 2,4-D and decapitated seedlings were painted with the natural auxin IAA with or without an inhibitor of DNA synthesis, FUdR. All treatments resulted in massive swelling at the pea apex because of cell expansion cell divisions also occurred, but not in the presence of FUdR (6). Cellulases were extracted as described in Figure 1 and assayed in unpurified form.

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