Stanley H. Duke

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

Starch-degrading enzymes in isolated pea (Pisum sativum L. cv. Laxtons Progress No. 9) chloroplasts were investigated and compared with those in crude pea leaf and stipule preparations. End-product analysis of amylopectin degradation by chloroplast and crude extracts indicates that maltose is the major product of both. Two multiforms of β-amylase (EC 3.2.1.2) were detected in pea chloroplasts using an electrophoretic transfer technique. A starch-debranching enzyme (EC 3.2.1.10) was detected in chloroplasts by electrophoretic transfer and the degradation of pullulan. A different multiform of debranching enzyme was found in crude preparations. α-Amylases (EC 3.2.1.1) were detected by electrophoretic transfer through gels containing starch and starch azure, and by change in viscosity of a starch solution, but were only found in crude preparations indicating an extrachloroplastic location. Incubation of maltotriose with chloroplast extracts gave high levels of glucose production and formation of oligosaccharides with degrees of polymerization larger than that of maltotriose indicating transglycosylase (EC 2.4.1.25) activity. Neither α-glucosidase (EC 3.2.1.20) nor maltose-phosphorylase (EC 2.4.1.1) activity were found in either chloroplast or crude preparations, whereas starch-phosphorylase (EC 2.4.1.1) activity was in both. The possible role of these enzymes in starch degradation by pea chloroplasts is discussed.

Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

SummaryDiploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 107 suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.

The Role of Pea Chloroplast [alpha]-Glucosidase in Transitory Starch Degradation

Pea chloroplastic [alpha]-glucosidase (EC 3.2.1.20) involved in transitory starch degradation was purified to apparent homogeneity by ion exchange, reactive dye, hydroxylapatite, hydrophobic interaction, and gel filtration column chromatography. The native molecular mass and the subunit molecular mass were about 49.1 and 24.4 kD, respectively, suggesting that the enzyme is a homodimer. The enzyme had a Km of 7.18 mM for maltose. The enzymes maximal activity at pH 7.0 and stability at pH 6.5 are compatible with the diurnal oscillations of the chloroplastic stromal pH and transitory starch accumulation. This pH modulation of the [alpha]-glucosidases activity and stability is the only mechanism known to regulate starch degradative enzymes in leaves. Although the enzyme was specific for the [alpha]-D-glucose in the nonreducing end as the glycon, the aglycon moieties could be composed of a variety of groups. However, the hydrolysis rate was greatly influenced by the aglycon residues. Also, the enzyme could hydrolyze glucans in which carbon 1 of the glycon was linked to different carbon positions of the penultimate glucose residue. The ability of the [alpha]-glucosidase to hydrolyze [alpha]-1,2- and [alpha]-1,3-glucosidic bonds may be vital if these bonds exist in starch granules because they would be barriers to other starch degradative enzymes. This purified pea chloroplastic [alpha]-glucosidase was demonstrated to initiate attacks on native transitory chloroplastic starch granules.

A Comparison of Standard and Nonstandard Measures of Malt Quality

The objectives of this study were to compare standard and nonstandard measures of malting quality using simple correlations and to determine whether six highly elite malting barley cultivars could be distinguished from each other using multivariate statistics to analyze 9 standard and 22 nonstandard measures of malting quality. Simple linear regression revealed cultivar differences in the thermostabilities of α-amylase, β-amylase, limit dextrinase, and α-glucosidase that were positively correlated with differences in wort osmolyte concentrations (r = 0.853–0.958, P ≤ 0.05–0.01) and differences in the thermostabilities of α-amylase, β-amylase, and limit dextrinase that were correlated with diastatic power (r = 0.872–0.937, P ≤ 0.05–0.01). Principal component analysis (PCA) of the nonstandard measures of malting quality were considered more useful than PCA of the standard measures because the former was able to categorize the six-row cultivar and two-row cultivar with the lowest real degree of fermentation, an important measure of brewhouse performance, as being different from the other two- and six-row malts. The malt quality traits that distinguished the two lowest performing of the six elite malting barleys from the other malts were α-glucosidase, limit dextrinase, and α-amylase activities, which were lower in these two malts, plus the thermostabilities of α-amylase, β-amylase, and limit dextrinase and wort osmolyte concentrations, which were higher in these two malts.

Ultrastructural localization of urate oxidase in nodules of Sesbania exaltata, Glycine max, and Medicago sativa.

SummaryThe localization of urate oxidase (=uricase, E.C. 1.7.3.3) was determined cytochemically in nodules of Sesbania exaltata (Raf.) Cory, soybean (Glycine max [L.] Merr.) and alfalfa (Medicago sativa [L.]), using the precipitation of peroxide (produced during the oxidation of urate) by cerium chloride. Cerium perhydroxide reaction product was noted only in the microbodies, a localization consistent with biochemical fractionation studies on urate oxidase. Urate oxidase was present not only in the uninfected cells of the cortical tissue, but also in both infected and interstitial cells in the central tissue, suggesting that at least this enzyme of ureide metabolism is not confined to interstitial cells. Urate oxidase cytochemistry of nodules from alfalfa (Medicago sativa L.), an amide producer, also resulted in microbody staining but the microbodies were infrequently noted in cell profiles.

Osmolyte concentration as an indicator of malt quality

This study was conducted to test the hypothesis that malt osmolyte concentrations can be used as an indicator of barley malt quality. Barley seeds of four six-row and four two-row genotypes were steeped and then germinated for 6 days at 20°C. At intervals of 24 hr over the germination regime, green malt from each cultivar was removed, kilned, and analyzed for osmolyte concentration (OC), malt extract (ME), diastatic power (DP), α-amylase activity, soluble/total protein (S/T), and β-glucan concentration. OC increased most rapidly from days one to three of germination. After 4 days, rates of increase in OC began to slow and, after 5 days of germination, OC had plateaued or declined. In all but three genotypes, ME followed a pattern similar to that of OC, but ME levels plateaued or declined approximately 1 day sooner than OC. This suggests that OC continues to measure storage compound degradation for a longer period than ME and could be a better indicator of malt modification than ME. ME and OC were significantly and positively correlated in days two through four and day six (r = 0.740–0.927, P < 0.0001). For days two and three, OC correlated well with ME for all days (r = 0.740–0.942, P < 0.0001) and α-amylase activity for day two (r = 0.771, P < 0.0001). For day two, OC correlated well with days two through six for β-glucan concentration (r = −0.702 to −0.830, P < 0.0001). No significant correlations were found for DP and OC on any day. These data indicate that OC of malt produced at early time points in germination is a good indicator of several measures of the quality of malt produced at later time points in germination.

Differential RNA expression of Bmy1 during barley seed development and the association with β-amylase accumulation, activity, and total protein.

The objective of this study was to determine if developing barley (Hordeum vulgare L.) seeds had differences in β-amylase 1 (Bmy1) mRNA accumulation, β-amylase (EC 3.2.1.2) activity, β-amylase protein accumulation, and total protein levels during late seed development from genotypes with different Bmy1 intron III alleles. Two North American malting barley cultivars (Hordeum vulgare ssp. vulgare) were chosen to represent the Bmy1.a and Bmy1.b alleles and, due to limited Bmy1 intron III allele variation in North American cultivars, two wild barleys (Hordeum vulgare ssp. spontaneum) were chosen to represent the Bmy1.c and Bmy1.d alleles. Wild barleys Ashqelon (Bmy1.c) and PI 296897 (Bmy1.d) had 2.5- to 3-fold higher Bmy1 mRNA levels than cultivars Legacy (Bmy1.a) and Harrington (Bmy1.b). Levels of Bmy1 mRNA were not significantly different between cultivated or between wild genotypes. In all four genotypes Bmy1 mRNA levels increased from 17 to 19 days after anthesis (DAA) and remained constant from 19 to 21 DAA. Ashqelon and PI 296897 had more β-amylase activity on a fresh weight basis than Legacy and Harrington at all developmental stages. β-Amylase protein levels increased from 17 DAA to maturity in all genotypes. Total protein in grains from wild genotypes was significantly higher than cultivated genotypes at all developmental stages. Higher levels of total protein in Ashqelon and PI 296897 could explain their higher levels of β-amylase activity, when expressed on a fresh weight basis. When β-amylase activities are expressed on a protein basis there are no statistical differences between the wild and cultivated barleys at maturity.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

In search of a biological hour

Summary We focus here on a prominent subset of rhythms within the ultradian domain that have periods between 30 and 240 min as a candidate for a «biological hour.» Smooth histograms produced from reported periods obtained from two surveys of literature display prominent peaks centered near 95 min, with a range from about 50 to 130 min. The list of variables having these periodicities is extensive and diverse (e.g., circumnutation movements of plants and the REM/non-REM sleep cycle of human beings). The periodicity displayed by many of these examples is somewhat irregular, perhaps because of other changing biological components and/or environmental factors such as stress. For example, results from our experiments showed that when Phaseolm vulgaris L. shoots were subjected to stress caused by staking, the ultradian periodicity of circumnutation was initially lengthened and later restored to its «normal» length.

Metabolic changes in Avena sativa crowns recovering from freezing.

Extensive research has been conducted on cold acclimation and freezing tolerance of fall-sown cereal plants due to their economic importance; however, little has been reported on the biochemical changes occurring over time after the freezing conditions are replaced by conditions favorable for recovery and growth such as would occur during spring. In this study, GC-MS was used to detect metabolic changes in the overwintering crown tissue of oat (Avena sativa L.) during a fourteen day time-course after freezing. Metabolomic analysis revealed increases in most amino acids, particularly proline, 5-oxoproline and arginine, which increased greatly in crowns that were frozen compared to controls and correlated very significantly with days after freezing. In contrast, sugar and sugar related metabolites were little changed by freezing, except sucrose and fructose which decreased dramatically. In frozen tissue all TCA cycle metabolites, especially citrate and malate, decreased in relation to unfrozen tissue. Alterations in some amino acid pools after freezing were similar to those observed in cold acclimation whereas most changes in sugar pools after freezing were not. These similarities and differences suggest that there are common as well as unique genetic mechanisms between these two environmental conditions that are crucial to the winter survival of plants.