Fructan Content and Fructosyltransferase Activity during Wheat Seed Growth

Abstract

The objective of this research was to determine the changes in fructan content and the activity of fructosyltransferases during the growth of wheat seeds (Triticum aestivum L. Thell, cv Caldwell). The total fructan content of the seeds decreased significantly during seed growth. The trisaccharide and tetrasaccharide content increased from 6 to 28 days post anthesis (DPA) and then declined, but these changes are notstatis-tically significant. The content or concentration of longer chain polymers did decline significantly (64.55-6.52 milligrams per gram dry weight). Free fructose also decreased significantly during seed growth indicating that the fructose liberated from the decrease in fructan content was utilized by the seed. Sucrose increased significantly from 6 to 12 DPA, then declined significantly from 12 to 28 DPA. Sucrose:sucrose fructo-syltransferase activity was greatest from 6 to 12 DPA (averaging 0.16 micromole of fructose transferred per seed per hour), then declined rapidly (0.04 micromole of fructose transferred per seed per hour). The estimated activity of fructan:fructan fructosyltransferase followed a similar pattern. The increase in sucrose concentration and high enzyme activity suggests that fructans were synthesized during the lag phase of seed growth. Wheat species (Triticum sp.), like other grasses of Poales, accumulate polymers of fructose called fructans. They have been isolated in stems (1, 6), leaves (23), and seeds (2, 4). Of particular interest here is the kernel or seed accumulation of fructans. The quantity of fructan in the seed has been reported to decline during growth from about 1.0 to 0.25 mg per seed (2, 4). It is not clear whether the initial fructan content of the seed results from accumulation before anthesis or from synthesis during seed growth. Fructans are believed to be synthesized by SST2 and FFT. SST transfers a fructose moiety from one sucrose molecule to another producing a trisaccharide and glucose. FFT catalyzes the synthesis of higher order polymers. The research published on SST indicates the only substrate is sucrose since UDP-fructose does ' not act as a substrate (17, 18). Second, localization ofthe enzyme suggests it is exclusively vacuolar (5, 22). The pH optimum ranges from 5.0 to 5.6 (19). The correlative changes in SST activity and fructan synthesis, and the fact that it catalyzes the synthesis of the first fructan (the trisaccharide) suggests SST controls carbon partitioning into the fructan pool (18, 19). The enzyme synthesizing fructan polymers beyond the trisac-charide is FFT. Little is known about this enzyme. Much ofwhat is known has been deduced from work on Jerusalem artichoke (Helianthus tuberosus) or dandelion roots (Taraxacum officin-ale) (19). The pH optimum of FFT is higher (6.1-8.5) than that of SST. Both dandelion and Jerusalem artichoke produce inulin-type fructans. Lolium (11) and wheat blades (22) are the only Gramineae tissue for which FFT activity estimates have been made. In wheat blades the tetrasaccharide was synthesized in cell-free extracts, although higher-order polymers were extracted from the tissue (22). The objectives of our study were to examine the changes in fructan content and the enzymes of fructan synthesis during maturation of wheat seeds. We have been studying carbon accumulation and metabolism in wheat seeds and noticed the presence of fructans in developing kernels. We were interested in estimating both the changes in fructan content and the potential for synthesis of fructan during seed growth. MATERIALS AND METHODS Plant System. A soft red winter wheat cultivar (Triticum aestivum L. em Thell., cv Caldwell), was grown in the field at the Purdue University Agronomy Farm (West Lafayette, IN) in a randomized complete-block design. Each plot contained four 2.5 m rows which were spaced 30 cm apart. The seeding rate was 1 seed per cm. The time of anthesis was considered to be when anthers first became visible in the spike. Individual spikes were tagged at anthesis and harvested at 2 to 4 d intervals from anthesis until maturity. Spikes were frozen in liquid N2, placed in plastic bags and transferred on dry ice to a-80°C freezer until used. Grains from the central spikelets were weighed, lyophilized, and reweighed to obtain fresh weight, dry weight and water content measurements. Data presented are the means of 8 rep-lications. Separation of Fructans. Grains were extracted with 80% ethanol (v/v) and water to obtain sugars. The sugar extractions were flash evaporated and reconstituted with distilled H20. Sugars were separated by passing the solution through a 110 x 1.5 cm column of Bio-Gel P2 (200-400 mesh). Aliquots were collected by elution with 4 g/L NH4HCO3 and fractions were assayed colorimetrically for free and combined fructose by reaction with anthrone (13). The optical density obtained by reaction of fructose standards with anthrone was utilized to determine the fructose equivalents of the separated sugar fractions. The free https://plantphysiol.org Downloaded on April 29, 2021.-Published by