Extensive degradation of protein during fermentation of high‐protein crops reduces efficiency of dietary N utilization in ruminants. Evidence suggests that enhanced levels of fermentable carbohydrates can reduce proteolysis. Our objective was to evaluate whether delaying daily cutting time, to allow total nonstructural carbohydrates (TNC) to accumulate, would inhibit protein degradation by way of greater acid production in the silo. Red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) were harvested at 0600, 1000, 1400, and 1800 h in 1993, 1994, and 1995 and wilted to a dry matter (DM) content of 350 g kg−1 before ensiling. The level of TNC in fresh forage of both species increased throughout the day. Starch accounted for most of the daily change in TNC in fresh alfalfa, whereas in red clover, quantitative increases in sugar and starch impacted TNC similarly. Level of TNC at initiation of ensiling did not consistently affect protein degradation during fermentation as confirmed by generally insignificant correlation coefficients. The extent of proteolysis in the silo was consistently greater in alfalfa than red clover. Silage pH typically decreased and starch increased as cutting time was delayed from 0600 to 1800 h. While the extent of proteolysis was largely unaffected by inherent increases in TNC, lower silage pH and higher starch concentrations indicate that silage from the afternoon cuttings may be better preserved and higher in quality.

trolling these traits interact with each other and variable environmental stresses to determine winter hardiness. Winter hardiness is a complex trait and one of the most importantThe severe winters necessary to evaluate winter hardiadaptations for alfalfa (Medicago sativa L.) grown in northern cliness only occur every 3 to 4 yr, on average, in the upper mates. In the absence of winter hardiness data, alfalfa breeders predict the potential of genotypes with component traits related to winter Midwest (Shaeffer et al., 1992) further complicating hardiness. This research was undertaken to identify and compare breeding for winter hardiness. some of the genomic regions that control winter injury (WI) and twoIn the absence of direct measurements of winter harcomponent traits, fall growth (FG) and freezing injury (FI). Two diness, the winter hardiness potential of genotypes and plants, B17 and P13, representing the extremes for each trait were cultivars has been predicted from related traits that are crossed, and a F1 plant was backcrossed to each parent to create two believed to represent components of winter hardiness. populations of 101 individuals each. Each population was scored forFall dormancy has traditionally been the primary com-82 single dose restriction fragment loci, and 17 or 19 two-allele loci ponent used to predict winter hardiness in alfalfa. Short and evaluated for FG, FI, and WI in 2 yr of replicated field trials. days and cool temperatures trigger the fall dormancy Trait measures over the 2 yr were significantly correlated (r ϭ 0.71, response (Nittler and Gibbs, 1959), which can be scored r ϭ 0.42, and r ϭ 0.76 for FG, FI, and WI, respectively). Significant easily by measuring vertical regrowth following the last correlations also existed between WI and FG (r ϭ 0.50 and 0.56) and FI (r ϭ 0.34 and 0.58) for each year. One to six single dose restriction

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbibition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose--phosphate dehydrogenase) or and that, with removal of the testa from seeds, the "leakage phenomenon" is enhanced (14,32). The amount of leakage during imbibition has been shown to correlate negatively with viability in studies with seeds of soybean (6, 39), pea (14,24), bean (17), and peanut (1) and has suggested to some that the leaked substances may, in some way, decrease viability. Another study has shown that removal of the testa of pea seeds results in death of the outer layers of cotyledonary cells during imbibition (25). The question arises: does the testa protect against leakage or is the leakage only a symptom of a fundamental dysfunction which can occur in imbibition?Two hypotheses have been promoted to explain the mechanism(s) ofleakage of solutes during the imbibition ofseeds. Larson (14) has suggested that cell membranes are ruptured during the initial phases of imbibition. Simon (31, 33) has proposed that the membranes of dry seeds are formed into hexagonal plates with pores formed in the areas of the phospholipid heads through which low-molecular weight solutes can leak from cells by passive diffusion during initial stages of membrane hydration (e.g. before phospholipids form typical bilayer membranes). Recently Powell and Matthews (25) have suggested that, in peas, cellular rupture and leakage through membranes may both occur when the testae are removed from seeds. To date, there has been a paucity of data that any macromolecules could move through the cell membrane during imbibition. Here, we have examined the leakage of imbibing seeds with and without testae for the presence of cytoplasmic, organelle, and organelle marker enzymes which would not pass through small membrane pores but which could only pass through very large membrane discontinuities or which would be the result of membrane rupture. In this way, we have tested both of the aforementioned hypotheses in a more definitive manner than has been hitherto reported.In the development of the legume seed, the testa appears to function in interconverting amino acids and sugars supplied by the phloem to the developing embryo (19,35,36) and in preventing injury by differentiating into a sclerified integument as the embryo matures (27). It has also been proposed that the testa protects seeds against "leakage" of intracellular substances during imbibition (32). This function has been suggested to be of great importance in the initial stages of germination of legume seeds in that many substances which leak from seeds may offer a substrate for potential pathogens (32). Past studies have demonstrated that electrolytes, sugars, amino acids, organic acids, and proteins are released from seeds during imbibition (1,6, 14,18,23,29,33,34) Mammoth Virgi...

The most abundant ,B-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this ,#-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a ,#-amylase by end product analysis and by its inability to hydrolyze #-limit dextrin and to release dye from starch azure. Pea #B-amylase is an approximate 55 to 57 kilodalton monomer with a pi of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a Km of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromercuriphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea ,B-amylase is competitively inhibited by its end product, maltose, with a K, of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with a-cyclohexaamylose being a stronger inhibitor than jl-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea jB-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea j-amylase is the multichain type.Possible roles of pea ,-amylase in cellular glucan metabolism are discussed.ofthese enzymes are located in the same cellular compartment as is particulate starch. Several studies with pea indicate that most of the cell's 3-amylase activity is extrachloroplastic and that chloroplasts contain very low or no fl-amylase activity (12, 13, and refs. contained therein). In contrast, one study with pea (34) indicates relatively high f,-amylase activity in chloroplasts; however, even in this study most of the f3-amylase was found to be extrachloroplastic with 50 to 60% localized in vacuoles. As higher plant particulate starch is contained within plastids, the role of vacuolar f3-amylase in starch degradation is uncertain. Furthermore, the role of nonvacuolar ,B-amylase has not been established, and in some storage tissues f3-amylase appears to be inessential for starch degradation (29).To understand the possible roles f3-amylase may have in cellular glucan metabolism, it is necessary to elucidate the physical and kinetic properties of the enzyme. While the characteristics of,B-amylase from storage tissues such as barley (15) and wheat (30) grains, soybean seeds (17, 19), and sweet potato tubers (29) have been well documented, much less information is known about ,3-amylases from tissues containing transitory starch, such as leaves. We present here the purification and characterization of,3-amylase fr...

The specific measurement of a-amylase activity in crude plant extracts is difficult because of the presence of f8-amylases which The specific determination of a-amylase activity in crude plant extracts is difficult because of the presence of 8-amylase activity in these tissues that directly interferes with most assay methods. The most commonly used procedure involves the selective inactivation of ,B-amylase by heating. This procedure is described by Briggs (4) and is based on the original observations of Schwarzer (23) 3 To whom reprint requests should be addressed.tivated by heating the malt to 70°C for 20 min in the presence of Ca2". a-Amylase, which is heat stable in barley malt, is then assayed by reducing power production or starch-iodine color disappearance. Whereas this procedure is used routinely in the malting industry, it has also been applied to tissues and plant species where the heat stabilities of the constitutive a-and ,Bamylases are unknown (1,5,12, 24). In fact, several investigators have shown that in stveral plant species, a-amylases are heat labile under these conditions (1,17,18).Another procedure involves the putative selective inactivation of fi-amylases by the addition of low concentrations of HgC12, (10)(11)(12). This method is clearly dependent on the selective HgCl2 inhibition of ,B-amylase, a premise that has not been adequately tested.A clinical method for the determination of a-amylase was developed by Rinderknecht et al. (21), using a chromogenic substrate specific to a-amylase. This substrate is potato starch derivatized with RBB4 commercially available as starch azure or amylopectin azure. This insoluble substrate is suspended in buffer and e-amylase action results in the solubilization of colored fragments of the starch azure. After the assay is terminated, the unreacted substrate is removed by filtering or by centrifugation and the color in solution is used as an estimation of the a-amylase activity. This method was originally developed for use in health sciences, and because animals have no ,B-amylase, interference by ,B-amylase was not considered. The use of this and similar chromogenic substrates for the determination of a-amylase activity (25) has received widespread use in clinical applications; however, this method has received only occasional use in the plant sciences (6,13,14,20). Previous plant studies using this assay have generally assumed that this substrate is specific for a-amylase and that ,8-amylase is not reactive. Bilderbach (3) demonstrated that the ,8-limit amylopectin azure, generated by the digestion of amylopectin azure with ,B-amylase, would release no color by further treatment with ,B-amylase, but would release color upon treatment with a-amylase. He also found that the presence offi-amylase had an interfering effect on the release of color from this substrate by a-amylase, but did not characterize this in detail. He presented the procedure as a qualitative rather than quantitive procedure for a-amylase determination. Apparently, no further ...