Microsomal flavonoid 3'-monooxygenase from maize seedlings

Identification of flavonoid 3′-monooxygenase establishes another reaction in the biosynthesis of flavonoid compounds in maize (Zea mays L.). The flavonoid 3′-hydroxylase was obtained as a microsomal enzyme preparation by buffer extraction of 5 day old maize seedlings and ultracentrifugation. Seedlings were exposed to light 24 hours prior to enzyme extraction. The extraction buffer required the addition of sucrose or glycerin and dithiothreitol to obtain an active hydroxylase that retained its activity on storage at −70°C. Enzymic activity required O₂ and NADPH, was optimum at pH 8.5 and 30°C, and could be inhibited 79% by carbon monoxide. Carbon monoxide inhibition could be reduced to 21% by irradiation of the samples with 450 nanometer light during incubation. Kaempferol, a flavonol; naringenin, a flavanone; and apigenin, a flavone, all served as substrates for the hydroxylase. Treatment of the microsomal enzyme preparation, previously reduced with sodium dithionite, with carbon monoxide gave a 455 nanometer absorption peak which disappeared on oxidation of the preparation with the formation of a 420 nanometer peak. These results suggest a cytochrome P-450 type monooxygenase enzyme. The concentration of cytochrome P-450 was 0.21 nanomoles per milligram protein. Identification of the monooxygenase provides further biochemical information about a biosynthetic sequence for which the genetics have been studied intensely.     

Levels of Free and Conjugated Abscisic Acid in Developing Floral Organs of the Navel Orange (Citrus sinensi [L.] Osbeck cv Washington)

The levels of abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate (putatively identified as the glucosyl ester, abscisyl-β-d-glucopyranoside) were determined by enzyme immunoassay in the organs of developing navel orange (Citrus sinensis [L.] Osbeck cv Washington) flowers. Although both compounds were detected in every tissue, developmentally related differences between organs in the total and relative contents were observed. The highest ABA levels were observed in the stigma/style shortly after anthesis (11.5 ± 0.6 nanomoles ABA per gram fresh weight and 4.8 ± 0.6 nanomoles ABA-conjugate per gram fresh weight); whereas, the highest ABA-conjugate levels were observed at the same time in the floral disc (hypogynous disc plus calyx; 3.5 ± 0.1 nmol nanomols ABA per gram fresh weight and 11.8 ± 0.9 nanomoles ABA-conjugate per gram fresh weight). These results suggest that differences in ABA content reflect tissue-specific variation in the facility for ABA conjugation. Increased ABA levels were observed in the stigma/style near anthesis; however, a relationship with pollination is discounted, since `Washington' navel orange flowers are male sterile and devoid of pollen.     

Cyanobacterial Acclimation to Photosystem I or Photosystem II Light

The organization and function of the photochemical apparatus of Synechococcus 6301 was investigated in cells grown under yellow and red light regimes. Broadband yellow illumination is absorbed preferentially by the phycobilisome (PBS) whereas red light is absorbed primarily by the chlorophyll (Chl) pigment beds. Since PBSs are associated exclusively with photosystem II (PSII) and most of the Chl with photosystem I (PSI), it follows that yellow and red light regimes will create an imbalance of light absorption by the two photosystems. The cause and effect relationship between light quality and photosystem stoichiometry in Synechococcus was investigated. Cells grown under red light compensated for the excitation imbalance by synthesis/assembly of more PBS-PSII complexes resulting in high PSII/PSI = 0.71 and high bilin/Chl = 1.30. The adjustment of the photosystem stoichiometry in red light-grown cells was necessary and sufficient to establish an overall balanced absorption of red light by PSII and PSI. Cells grown under yellow light compensated for this excitation imbalance by assembly of more PSI complexes, resulting in low PSII/PSI = 0.27 and low bilin/Chl = 0.42. This adjustment of the photosystem stoichiometry in yellow light-grown cells was necessary but not quite sufficient to balance the absorption of yellow light by the PBS and the Chl pigment beds. A novel excitation quenching process was identified in yellow light-grown cells which dissipated approximately 40% of the PBS excitation, thus preventing over-excitation of PSII under yellow light conditions. It is hypothesized that State transitions in O₂ evolving photosynthetic organisms may serve as the signal for change in the stoichiometry of photochemical complexes in response to light quality conditions.     

Photocontrol of Dark Circadian Rhythms in Stomata of Phaseolus vulgaris L

Stomatal diffusion resistance in primary leaves of Phaseolus vulgaris L. which had been grown in light:dark cycles followed a marked circadian rhythm when the plants were transferred to continuous darkness. Reentrainment of the rhythm required more than one inductive change in photoperiod. The phasing of the rhythm of dark stomatal opening was contolled primarily by the light-on (dawn) signal, whereas the rhythm of dark closure was related to the light-off (dusk) signal. The evidence points to a dual control of the circadian clock in which a product of photosynthesis plays a major role. No evidence for phytochrome involvement in the phasing of the rhythm was found. An influence of phytochrome on the amplitude of the stomatal rhythm was observed in which removal of phytochrome-far-red absorbing form caused rapid damping.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Effects of Atmospheric CO(2) Enrichment on the Growth and Mineral Nutrition of Quercus alba Seedlings in Nutrient-Poor Soil

One-year-old dormant white oak (Quercus alba L.) seedlings were planted in a nutrient-deficient forest soil and grown for 40 weeks in growth chambers at ambient (362 microliters per liter) or elevated (690 microliters per liter) levels of CO₂. Although all of the seedlings became severely N deficient, CO₂ enrichment enhanced growth by 85%, with the greatest enhancement in root systems. The growth enhancement did not increase the total water use per plant, so water-use efficiency was significantly greater in elevated CO₂. Total uptake of N, S, and B was not affected by CO₂, therefore, tissue concentrations of these nutrients were significantly lower in elevated CO₂. An increase in nutrient-use efficiency with respect to N was apparent in that a greater proportion of the limited N pool in the CO₂-enriched plants was in fine roots and leaves. The uptake of other nutrients increased with CO₂ concentration, and P and K uptake increased in proportion to growth. Increased uptake of P by plants in elevated CO₂ may have been a result of greater proliferation of fine roots and associated mycorrhizae and rhizosphere bacteria stimulating P mineralization. The results demonstrate that a growth response to CO₂ enrichment is possible in nutrient-limited systems, and that the mechanisms of response may include either increased nutrient supply or decreased physiological demand.     

Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion

An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme-linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function.     

Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures

Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6β-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6β-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe²⁺ ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-¹⁴C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO₂ from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6β-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.     

Sucrose synthase activity in developing wheat endosperms differing in maximum weight

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the “rate limiting” enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.     

Phycomyces: fine structure analysis of the growing zone

Fine structure analysis of the stage IVb Phycomyces sporangiophore growing zone (GZ) was performed during steady-state growth using a computer-video digitizer and recorder. By simultaneously measuring the trajectory of two independent particles above and within the GZ, we have confirmed the previous findings of R. Cohen and M. Delbrück (1958 J Cell Comp Physiol 52: 361-388) that the GZ is not uniform. We have been unable to confirm their findings that counterclockwise rotation exists in a mature sporangiophore. The rates of rotation and elongation change independently as a function of position in the GZ. This change is not linear as would be expected if the GZ were uniform. The importance of this finding is discussed in terms of the fibril reorientation model.