Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

A role for nitrogen reserves in forage regrowth and stress tolerance

Carbohydrate accumulation and utilization during shoot regrowth after defoliation and winter has been studied extensively in most species used as forage. However, recent work suggests that N reserves found in vegetative tissues also are important for defoliation tolerance and winter hardiness. Results suggest that these N reserves constitute an alternative N source used when N₂ fixation and/or mineral N uptake are reduced. ¹⁵N labelling experiments indicate that a large proportion of herbage N is derived from N reserves mobilized from stem bases or roots to developing leaves and shoots. Amino acids and specific proteins (i.e. vegetative storage proteins, VSPs) are deposited in roots and stem bases and, in the case of VSPs, are degraded rapidly after defoliation. Identification and characterization of VSPs will increase our understanding of the role N reserves play in stress tolerance and may lead to innovative approaches for improving forage persistence and productivity.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Carbohydrate metabolism in leaf meristems of tall fescue : I. Relationship to genetically altered leaf elongation rates

The physiological bases for genetic differences in leaf growth rates were examined in two genotypes of tall fescue (Festuca arundinacea Schreb.) selected for a 50% difference in leaf elongation rate. Genotypes had similar dark respiration rates and concentrations of carbohydrate fractions in the leaf meristem and in each daily growth segment above the meristem. Dark respiration rates and concentrations of nonreducing sugars, fructans, and takadiastase-soluble carbohydrates were highest in leaf intercalary meristems and declined acropetally with tissue age. Concentrations of reducing sugars were 1.0% of dry weight in leaf meristems, 3.7% of dry weight in tissue adjacent to the meristem, then decreased progressively with distance from the meristem. Glucose, fructose, and myo-inositol comprised over 90% of the monosaccharides present in leaf meristems. Soluble protein concentration was 9.7 milligrams per gram fresh weight in leaf meristems, 5.5 milligrams per gram in tissues immediately above the meristem and, thereafter, increased linearly with distance from the meristem.

Leaf meristems of the genotype exhibiting rapid leaf elongation contained 30% more soluble protein than those of the genotype selected for slow leaf elongation. The 4-fold difference in size of the leaf meristem appeared to be more important in influencing leaf elongation than were other characteristics examined.

     

Soybean shoot and root response to localized water and potassium in a split-pot study

Potassium- (K) and water-stratification in conservation tillage, rain-fed agroecosystems may reduce soybean [Glycine max (L.) Merr.] performance. A split-pot experiment with two soil-K levels [80 mg Kg^?1 (Low-K) and 164 mg Kg^?1 (Optimum-K)] and two soil-water contents [insufficient (Dry): variable between 55 to 85% field capacity (FC); sufficient (Wet): constant at 85 to 95% FC] was imposed to determine the effect of synchronous and asynchronous availability of localized K and soil water on soybean roots, nutrient uptake, and shoot growth. Asynchrony of soil water and K had no impact on soybean dry matter accumulation or nutrient uptake. Optimum soil K levels were relatively more important than soil water content to increase K availability and K tissue concentration and accumulation. Shoot?C and root-growth responded more to water?C than to K-stress. Shoot-K accumulation increased concomitantly with sufficient water availability and greater root surface area (RSA), but RSA was more important relative to water for K accumulation. Optimum-K with sufficient-water increased K accumulation by 50% compared to the insufficient-water treatment. Since enhanced K uptake occurred with greater RSA, and roots proliferated in response to water availability and not localized K, K should be placed in the soil fraction that provides sufficient water availability.     

Composition and Structure of Starch from Taproots of Contrasting Genotypes of Medicago sativa L

The objective of this study was to examine the composition and branch chain lengths of alfalfa (Medicago sativa L.) taproot starch during starch utilization and reaccumulation in response to defoliation. Genotypes were propagated vegetatively and well-established plants were sampled at defoliation and at weekly intervals thereafter. Starch granules from root tissues were dispersed in dimethyl sulfoxide and starch components separated using gel permeation chromatography. Root starches also were debranched enzymically, and branch chain lengths were examined. Results indicate that, irrespective of starch concentration, starch from taproots of the high starch genotype was composed of approximately 80% high molecular weight starch with I₂-Kl absorbance characteristics similar to amylopectin. The remaining 20% of the starch was low molecular weight with I₂-Kl absorbance characteristics similar to amylose. Starches of the low starch genotype contained approximately 85% high molecular weight polysaccharide at high root starch concentrations (>50 grams per kilogram). At low root starch concentrations (<10 grams per kilogram), starch from the low starch genotype had nearly equal proportions of low and high molecular weight polysaccharide. The I₂-Kl absorbance properties of the low molecular weight starches from roots of the low starch genotype indicated that some branching may be present. The distribution of chain lengths from amylopectin did not change during starch degradation and reaccumulation for the high starch genotype. In the low starch genotype, the proportion of low molecular weight branches having a degree of polymerization between 1 and 30 was decreased at the very low starch concentrations observed on the 14th day of regrowth. Higher concentrations and/or quantities of starch in roots of the high starch genotype were not associated with greater rate of herbage regrowth, when compared to the low starch genotype.     

Radioprotective effect of inosine and its enhancement by magnesium and global hypoxia.

The slight radioprotective action of inosine, when injected intraperitoneally to mice shortly before gamma-irradiation, can be enhanced by the administration of magnesium aspartate. This effect can be explained by the additivity of the vasodilatory actions of both agents. Inosine increases the radioprotective effectiveness of hypobaric hypoxia (10% O2), probably due to the additivity of the hypoxic effects in radiosensitive tissues. Acute hypoxic toxicity, however, is decreased by inosine administration. The cumulation of radioprotective effects of inosine and of its antihypoxic action in vitally important organs can have a favourable influence in hypoxic radiotherapy.     

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.