Distribution of a - Galactosidase in Cucurbita pepo

The distribution of a-galactosidase (a-D-galactoside galactohydrolase IEC 3.2.1.221) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series (la-1-6-galactopyranosyllj sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of agalactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total a-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl olHgosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl olHgosaccharides between the phloem and surrounding tissues in this plant. Members of the raffinose family of oligosaccharides, (a1-6-0galactopyranosyl). sucrose, are widely distributed in the higher plant kingdom with the lowest mol wt member of the series, raffmose, occurring most frequently (7). They commonly appear among the soluble storage products of seeds (1, 16) and roots (15, 16) and have been reported in leaves (15). In many plant species their occurrence appears restricted to the seed where they are synthesized during maturation of the tissue and rapidly disappear during the initial stage of germination (1). During the past 2 decades, however, investigations of phloem translocation in a number of widely divergent species have established that these oligosaccharides may also accompany sucrose as major transport sugars in a number of plant families (23, 25). Studies of their metabolism have been mostly confined to seed tissue, where it is generally acknowledged that their enzymic hydrolysis is affected by a-galactosidase (a-D-galactoside galactohydrolase [EC.3.2.1.221) and considerable evidence for the occurrence and properties of this enzyme in germinating seed extracts has been accumulated (3). It would seem reasonable, therefore, to expect those plants that transport these oligosaccharides in their vascular tissue to possess similar a-galactosidase activities in their immature and developing organs, but the nature and function of a-galactosidase activity in plant tissues other than the seed have received scant attention. The enzyme has been briefly reported to be in 'This research was funded by National Research Council of Canada Grant A2827 to J. A. W. 2 Present address: Department of Botany, Plant Science Laboratories, University of Reading, Whiteknights, Reading, England RG6 2AS. 'To whom correspondence should be addressed. leaves (3, 8) where an involvement in galactolipid metabolism of organelle membranes was suggested, and it has also been reported in walls of epicotyl tissue (12) and in plant cell and tissue cultures (10, 13). Our interest in the distribution of a-galactosidase in Cucurbita pepo resulted from our discovery that immature importing leaves possess an extremely active ability to hydrolyze raffimose, stachyose, and verbascose entering their tissue from the phloem and are incapable of any detectable synthesis or accumulation of these sugars. In contrast, mature exporting leaves demonstrate both a synthesis and a negligible in vivo hydrolysis of these oligosaccharides (20, 22, 23). We hypothesized that an extremely active a-galactosidase system is operating in immature leaf tissue, while in mature leaves its activity drastically declines due either to loss or inactivation or, alternatively, through an effective compartmentation becoming established during leaf development. We reject the concept of loss because ofour subsequent discovery that extracts from mature leaves of C. pepo contain at least three active isozymic forms of a-galactosidase (18). Preliminary experiments indicated that the bulk of the a-galactosidase activity in mature leaf tissue is indeed compartmentalized in the cell wall (unpublished data) and this conforms with increasing evidence for restriction of glycosidase enzymes to cell walls (13). Before extending our studies of the intracellular location of agalactosidase activity we considered it of interest to discover whether the three isozymic forms are distributed in other tissues of C. pepo and to attempt to relate their presence to the utilization of the galactosyl oligosaccharides in these tissues. These results are reported here. MATERIALS AND METHODS Plant Material. Plants of the Early Prolific Straight-Neck Squash, Cucurbita pepo L. var. melopepo f. torticollis Bailey (W. A. Burpee, Philadelphia) were used in all experiments. Leaf blades, petioles, flowers, and roots were obtained from plants that had been germinated in Vermiculite and grown in Perlite in controlled environment cabinets (20). Blades and petioles were harvested when the plants were between 2 and 5 weeks old. The morphological age of the blades was determined by a leaf plastochron index (5), based on a petiole length of 30 mm (19). Between LPI4 0 and 0.6 the blades had expanded their surface areas from about 8 to 20% of their final size and during this period they demonstrated maximal rates of import. The transition from a totally importing to a totally exporting blade occurred during the interval between LPI 0.5 and 1.5 (50%o expanded). At LPI 0.9 only the basal half of the blade was importing. At LPI 2.5 (80% expanded) export from the blade approached its maximal rate. The blade was 100%o expanded at LPI 4.0 (19). Flowers were obtained as fully expanded buds prior to opening from 6to 7week-old plants and roots were obtained by excising the entire rooting system from 1-week-old seedlings. Seed extracts were prepared from whole seeds imbibed where appropriate on filter 4Abbreviation: LPI: leaf plastochron index; LI, LII, LIII: isozymic forms of a-galactosidase. 713 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 62, 1978 paper in glass Petri dishes at 27 C in darkness. Enzyme Preparations. For determination of total a-galactosidase activity source material was rapidly weighed and homogenized in a pestle and mortar using 2 volumes of 100 mm sodium phosphate buffer (pH 7.0) in the presence of acid-washed silver sand. The extract was transferred quantitatively to a centrifuge tube with a further 2 volumes of buffer solution and centrifuged at 25,000g for 30 min at 2 C. The supernatant was assayed for agalactosidase activity. During centrifugation of seed extracts a lipid layer collected on the surface of the supernatant and a Pasteur pipette was used to remove the clear supernatant from below. For preparation of the isozymes the crude buffer extracts obtained from source material as described above were treated with solid ammonium sulfate and the protein fraction precipitating in the 30 to 60%o range was collected by centrifugation (18). The precipitate was redissolved in 2 ml of extraction buffer and after centrifuging at 10,000g for 10 min at 2 C, the supernatant was passed through a column of Sephadex G-100 (85 x 1.5 cm) and eluted with 100 mm sodium phosphate buffer (pH 7.0), flow rate 0.25 ml/min at 2 C. Fractions of 5 ml were collected and each was assayed for a-galactosidase activity. For all tissues examined activity was confined to a single peak which emerged from the column at a constant elution volume. The active fractions were pooled for each tissue and reduced in volume to 1.5 ml by ultrafiltration using an Amicon PM-10 membrane. Further purification of the a-galactosidase fraction was effected on a DEAESephadex A-50 column (30 x 1.5 cm) which had been equilibrated with 0.01 M NaCl in 100 mm sodium phosphate buffer (pH 7.0) at 2 C. The enzyme sample was run onto the column followed by 5 ml of the equilibration buffer. The column was then eluted at a flow rate of 0.2 ml/min with a linear gradient of 0.01 to 0.5 M NaCl in a total volume of 150 ml sodium phosphate buffer (pH 7.0) at 2 C. Fractions of 3 ml were collected and each fraction assayed for a-galactosidase activity. Enzyme Assays. a-Galactosidase activity was measured using p-nitrophenyl-a-D-galactopyranoside as the substrate (18). Assay mixtures contained 200 p1 of 3 mg/ml substrate in 200 mm sodium acetate buffer (pH 5.0) and 200 ,l of suitably diluted enzyme preparation in 100 mm sodium phosphate buffer (pH 7.0). After incubation at 35 C for 15 min the reaction was stopped by adding 2 ml of 5% Na2CO3 and the p-nitrophenol released was measured at 400 nm. Blanks were prepared by adding enzyme after Na2CO3. The unit of enzyme activity (U) was defined as the quantity that hydrolyzes 1 ,umol of substrate/min under the conditions outlined above. Total protein was measured using crystalline BSA as standard (1 1). RESULTS AND DISCUSSION a-Galactosidase Activty in Leaves. Our earlier results had shown that young leaves (LPI <0.3) rapidly hydrolyzed imported raffmose, stachyose, and verbascose, and they appeared incapable of synthesizing or accumulating these oligosaccharides (20, 23). Older leaves (LPI >1.5) by contrast were able to synthesize and accumulate these sugars (23) and if translocation from the blade was curtailed (e.g. by a petiole cold block) they appeared to demonstrate little metabolic turnover within this mature tissue (22). If the level of a-galactosidase activity is a controlling factor in this system our results may be explained through a depletion of total enzyme activity during leaf maturation or through an appreciable proportional shift among the three isozymic forms of which one or more is inactive or compartmentalized elsewhere in the cell. In the first series ofexperiments we assayed total a-galactosidase activity in the fifth leaf of C. pepo (19) at progressive developmental stages over a period of LPI 0 to LPI 2.5. Several leaves were taken at each LPI interval and the units of a-galactosidast activity measured per leaf were plotted againts the average fresh weight and the average total protein content per leaf (Fig. 1). Despite the age variability of the source material it is clear that a straight line relationship is obtained in both cases indicating that the level of a-galactosidase activity remains remarkably constant throughout leaf development to maturity. The proportion of the protein component of the leafs fresh weight soluble in the phosphate buffer does not alter over this period. In addition to measuring total a-galactosidase activity for whole leaves we also measured the distribution of activity between the proximal and distal regions of the fifth leaf at developmental stages between LPI 0.5 to 2.5. It is known that the physiological development of the leaf proceeds basipetally (19). Leaves were cut at right angles to the midrib to give approximately two equal halves. The halves were assayed for a-galactosidase activity separately and the specific activity values obtained from the two areas of the leaf compared (Table I). When assayed statistically by a paired t test procedure (21) the pairs of values were found to be not significantly different at the 5% probability level. This again supports the above evidence that the level of a-galactosidase activity, at least as measured in leaf extracts, does not alter during development of the blade from the immature to the mature stage. We also assayed the levels of total a-galactosidase activity in successive leaves on the same plant. The results in Table II were obtained from three plants where the 7th, 6th, and 5th leaves were at the importing, transitional, and exporting stages of physiological development, respectively (19). Again there is no significant evidence of a loss of total a-galactosidase activity from extracts of leaves during their maturation. The results also show that there is much less variability between leaves on the same plant than between equivalent leaves on different plants grown under identical conditions. The above values for total a-galactosidase activity presumably represented the combined in vitro activity of at least three isozymic forms of the enzyme (18). The physical form in which the enzyme exists may determine its activity in vivo and a distinction between