Isolation and characterization of isoperoxidases from Dee-Geo-Woo-Gen rice seedlings

Multiple forms of peroxidase from ten-day-old Dee-Geo-Woo-Gen (DGWG) rice seedlings were isolated by ammonium sulphate precipitation, polyacrylamide gel electrophoresis (PAGE) and DEAE-cellulose chromatography. The pH optima for P1-A and P1-B are 6.5–7.0 and 7.0 respectively. These isoperoxidases have apparently similar MWs and differ only in their electrophoretic and catalytic properties.     

Isolation,purification and characterization of adenosine triphosphatase from mitochondria of rice seedlings

A new and improved procedure is described for the isolation and purification of adenosine triphosphatase (F₁ ATPase) from rice seedling mitochondria. The enzyme has a multi-subunit structure, as revealed by electron microscopy and SDS-mercaptoethanol gel electrophoresis. Oligomycin sensitivity as well as cold lability of this enzyme clearly prove that the mitochondria of monocotyledons have an ATPase similar to the F₁ ATPases from other sources.     

Purification and characterization of diamine oxidase from rice embryos

Diamine oxidase of rice seedlings has been purified 1800-fold to homogeneity. The MW of the enzyme as determined by Sephadex G-100 gel filtration was 12.3 × 10⁴ and the enzyme contained two identical subunits each with a MW of 6.12 × 10⁴. The optimal temperature and pH for the enzyme were 30° and 7.5 respectively and the enzyme followed typical Michaelis kinetics with a K_m of 10^?5 M. Each enzyme molecule contained four molecules of FAD.     

Agmatine deiminase in rice seedlings

Agmatine deiminase activity in rice embryos increased gradually upto 24 hr during germination and then decreased. Gibberellic acid and kinetin inhibited the activity when added to the germination medium. The enzyme was purified 717 fold with specific activity 788.5 nkat/mg protein and yield 8.8%. The M_r of the native enzyme was 18.3 x 10⁴ and the enzyme was a dimer of two identical subunits. The pH and temperature optimum of the enzyme were 6.0 and 28° respectively. The enzyme followed typical Michaelis-Menten kinetics with a K_m value of 1.5 x 10^?2 M. The enzyme activity was inhibited by various divalent cations and spermidine and spermine, but putrescine showed no effect.     

Inactivation of nitrate reductase from wheat and rice leaves

In fresh leaves, the inactivation of nitrate reductase was rapid at high temperatures as compared to low temperatures. In leaves subjected to freeze-thaw treatment, the loss of enzyme activity was extremely rapid particularly at high temperatures. Pre-incubation with NADH not only protected the enzyme against inactivation, but also stimulated its activity. In dialysed extracts of rice leaves, NADH alone offered some protection while nitrate alone did not protect the enzyme from inactivation. Addition of both NADH and nitrate during pre-incubation enhanced the enzyme activity considerably. It is suggested that stimulation of nitrate reduction by NADH and nitrate may be of physiological significance to the plant, in the sense that in the presence of sufficient supplies of reluctant and nitrate, the process of nitrate assimilation would be accelerated.     

Properties of glutelin from mature and developing rice grain

Aminograms and SDS-polyacrylamide electrophoresis of milled rice glutelin of 12 Oryza sativa samples showed similar composition and ratio of 1 : 1 : 1 for subunits with MW 38 000:25 000: 16 000, indicating little possibility of finding variants of rice glutelins. Fractionation of S-cyanoethyl glutelin of 3 rices on polyacrylamide-agarose gels gave MW subunits differing in amino acid analysis of which the subunits with MW > 38 000 had the highest lysine content. Of the solubility fractions of endosperm glutelin, the fraction extracted by 0.5 M NaCl-0.6 % β-mercapto-ethanol-0.5% SDS was closest to glutelin in properties. In the developing grain of two varieties, appearance of protein bodies and rapid synthesis of glutelin from 7 days after flowering onward coincided with a drop in lysine content and appearance of MW 38 000 and 25 000 of crude glutelin. The MW 38 000 subunit is thus unique to endo-sperm glutelin.     

Properties of albumins of milled rice

Extraction studies on IR36 milled rice showed that albumins solubilized by 0.1–0.15 M (NH₄)₂SO₄ consisted of about 20% high(～5%) lysine, fast-migrating proteins on electrophoresis at pH 8.3 and about 80% lower ～2%) lysine proteins of slower mobility. The 2%-lysine albumins were insoluble in 1.8 M (NH₄)₂SO₄ while the higher lysine albumins required 4 M (NH₄)₂SO₄ to precipitate. The 2%-lysine albumins were not fractionated by gel filtration and gave only one major fraction with MW 19 000. SDS-polyacrylamide gel electrophoresis confirmed the major subunit to be of MW 17 000. These albumins were separated by DEAE-Sephacel chromatography at pH 8.5 into three fractions of similar aminograms but differing in analytical get electrophoretic and isoelectric focusing patterns.     

Fractionation of globulins of milled rice

Globulins were prepared by repeated precipitation with 1.3 M (NH₄)₂SO₄ from a 0.7 M NaCl extract of milled rice. Isoelectric precipitation at pH 4.5 did not effectively remove the α-globulin from the others. A major fraction that remained in solution during dialysis of the globulin precipitate against water was similar in properties to the globulin soluble at pH 4.5 during the isoelectric precipitation process. Some properties of this water-soluble globulin fraction are reported. Proteins extracted from milled rice at 50° with 0.5 M NaCl and precipitated as 1- to 3-μm particles on cooling were verified to be globulins.     

Growth and germination inhibitors in rice husks

A search for growth and germination inhibitors in rice husk (Oryza sativa L. cv Koshihikari) revealed four compounds, ineketone, S(+)-dehydrovomifoliol, momilactone-C and p-coumaric acid, in addition to the previously known momilactones-A and -B. The isolation and structural determination of these inhibitors are reported. The flavonoid tricin and three steroids were also detected in the husk but none showed any inhibitory activity.     

Isolation and characterization of two phytoalexins from rice as momilactones A and B

Two phytoalexins were isolated as chromatographically homogeneous amorphous solids from UV-irradiated, dark-grown rice coleoptiles. From their mass and ¹H NMR spectra, the compounds were characterized as the known diterpenes, momilactones A and B. The same compounds were also produced in blast-infected, WL 28325-treated rice leaves. They appear to be the first clearly identified cereal phytoalexins.     

Extraction and composition of rice endosperm glutelin

“Crude” glutelin was prepared from milled rice (Oryza sativa) flour by sequential extraction of the albumin-globulin fraction with 0.5 M NaCl and prolamin with 70% ethanol-0.6% β-mercaptoethanol. The solvent, 0.5% sodium dodecyl sulphate (SDS)-0.6% β-mercaptoethanol, extracted 91% of the endosperm glutelin without gelatinizing starch granules, whereas chaotropic solvents such as urea and guanidine caused extensive gelatinization. The S-cyanoethyl glutelin (Ce-glutelin) prepared by SDS extraction of the “crude” glutelin (9.5% protein) of IR480-5-9 rice gave three major subunits with MW 38000, 25000 and 16000 in the ratio 2:1:1 as determined by SDS polyacrylamide gel electrophoresis. A similar preparation from “crude” glutelin of a lower protein containing rice had the corresponding subunits in the ratio of 16:3:1. The MW 38000 subunit was unique to glutelin and was not present in C3-albumin-globulin or prolamin; the subunits were only partially purified by SDS Sephadex G-150 gel-filtration. The C3-glutelin was also prepared from a crude glutelin-prolamin preparation from IR480-5-9 by NaOH extractions followed by precipitation at pH 10 and ethanol extraction of the precipitate (C3-glutelin). This preparation had the same three major subunits and in the same ratio as C3-glutelin prepared by the SDS method. The subunits of the former preparation were separated by carboxymethyl Sephadex C-50 chromatography; the MW 38000 subunit eluted between pH 6.2–8.5, the MW 25000 in an impure state at pH values above 9, and the MW 16000 subunit was eluted at pH 8.6—9.2. Amino acid composition of the Ce-glutelin preparations were similar to each other. The MW 38000 and 16000 subunits had lower lysine contents than whole C3-glutelin, whereas the MW 25000 subunit had a higher lysine content.     

Properties of prolamin in mature and developing rice grain

A procedure was developed for preparing lipid- and phenol-free prolamin directly from IR480-5-9 milled rice (Oryza sativa L.).The preparation consisted mainly of one protein band on analytical and SDS-polyacrylamide disc gel electrophoresis with subunit MW of 17000 and a minor fraction with subunit MW 23000. The prolamin eluted as a single peak on SDS-Sephadex G-75 gel filtration and on DEAE-cellulose column chromatography. Prolamin was poor in lysine, histidine, cystine, and methionine but rich in glutamic acid, tyrosine and proline. In dehulled developing grain of two different rices, changes in the aminogram of the prolamin fraction coincided with the start of endosperm protein body synthesis and the appearance from 7 days after flowering of a second prolamin subunit with MW 23 000.     

Synergistic interaction of gibberellic acid and triazinone derivatives in the promotion of rice shoot growth

Forty-five 1,3,5-triazine-2,6-dione derivatives were synthesized and their plant growth-promoting activities examined by the rice (Oryza sativa) seedling test in the presence or absence of gibberellic acid (GA). For high activity in promoting the growth of rice seedlings and acting as active GA-synergists, a para-substituted or a 2,4-disubstituted phenyl group, a hydrogen atom and an alkoxy group were required in the 1-, 3- and 4-positions of the 1,3,5-triazine-2,6-dione molecule. 4-Ethoxy-1-(p-tolyl)-s-triazine-2,6(1H, 3H)-dione [TA], one of the most potent triazinones, synergized the effect of GA on the shoot elongation of different varieties of rice including normal type, dwarf mutants and chlorophyll-mutants. TA synergistically increased the growth-promoting activity of GA by both a simultaneous treatment at the same sites and separate treatments at separate sites of rice seedlings.     

Structural features of rice bran hemicellulose

Rice bran hemicellulose was fractionated by ammonium sulphate precipitation, ion-exchange chromatography and enzymatic techniques. Methylation analysis of each fraction revealed that the bran hemicellulose consisted mainly of highly branched arabinoxylan and xyloglucan. The acidic arabinoxylan main component appeared to have more doubly-branched xylose residues in the main chain and also more complicated side chains than the endosperm arabinoxylan. Xyloglucan was also isolated from the crude hemicellulose but the amount of β-(1 → 3), (1 → 4)-glucan was very small compared to the endosperm hemicellulose.     

Composition of cell wall preparations of rice bran and germ

Cell walls of petrol-defatted non-waxy IR32 rice bran and germ were prepared by protein removal with 0.5% SDS—0.6% β-mercaptoethanol, heating the residue to 80°, and destarching with Bacillus licheniformis α-amylase. A waxy rice, IR29, had a similar cell wall composition as IR32. Principal wall sugars were arabinose, xylose, and glucose. The 0.5 M sodium or potassium hydroxide and 8 M urea preferentially extracted arabinose-, xylose- and uronic acid-rich polysaccharides but 6 M sodium hydroxide—0.81 M boric acid extracted mannose-rich polysaccharides. DEAE-cellulose BO₃^3? chromatography of the 0.5 M sodium hydroxide extracts gave fractions of similar arabinose— xylose ratios. Proteins in the cell wall preparations had only 0.4–1.6% hydroxyproline, and were bound mainly to polysaccharides, based on disc gel electrophoresis. The preparations were autofluorescent in UV and rich in phenols, mainly ferulic acid. The cell wall preparations and their 8 M urea fractions had a softening effect on defatted waxy starch aqueous gel at 0.2–2% of the starch.     

Changes in salt-soluble proteins of rice during grain development

Salt-soluble proteins, albumin and globulin, were prepared from dehulled rice (Oryza sativa L., line IR1541-76-3) during grain development. Albumin and globulin progressively increased during grain development up to about 12 days after flowering (DAF) and then decreased slightly during grain desiccation. Free amino N was maximum at 10 DAF. Total protein and glutelin-prolamin (by difference) continued to increase up to 20 DAF. Aminogram of total protein and globulin showed a progressive decrease in lysine and threonine among the essential amino acids. Albumin showed a similar trend except for the lesser change in lysine content. Disc gel electrophoresis showed a maximum of four major and six minor protein bands for albumin and only one major and three minor bands for globulin. Sodium dodecyl sulfate-gel electrophoresis revealed three major polypeptide subunits for albumin with MW of 11 000, 8 500 and 16 000, and two for globulin with MW of 20 000 and 12 000.     

DNA topoisomerase I from rice: Enzyme synthesis in germination, and partial purification from cultured cells

DNA topoisomerase I activity was observed in two-day-old seeds of rice when the seeds started germination at 30°. Partially purified enzyme from cultured rice cells showed maximum activity at pH 7.0 with 75 mM NaCl in the absence of ATP, and showed resistance to camptothecin and DNA-intercalating reagents. The M_r was ca 80 000 using gel permeation on a Sephacryl S-200 column. After fractionation of the homogenate from cultured rice cells by centrifugation, the activity was observed mainly in the crude nuclear fraction.     

Covalently attached ferulic acid in a proteoglycan from rice bran

A water-soluble proteoglycan, precipitated with ammonium sulfate from the hot-water extract of rice bran, contained ferulic acid, which was liberated by alkaline treatment. Evidence for the linkage between the carboxyl group of ferulic acid and the proteoglycan was obtained by the characterization of ferulic acid hydroxamate after treatment of the proteoglycan with hydroxylamine.     

Coixlachryside A: A new lignan glycoside from the roots of Coix lachryma-jobi L. var. ma-yuen Stapf.

A new lignan glycoside, 1-[3,6-(4,4⿲-dihydroxy-3,3⿲-dimethoxy-δ-truxinyl)-β-fructofuranosyl]-α-glucopyranoside (1), named coixlachryside A, together with nine known simple phenolic compounds (3⿿11) were isolated from the roots of Coix lachryma-jobi var. ma-yuen using various chromatographic methods. Their chemical structures were determined based on the spectroscopic data interpretation, particularly circular dichroism (CD), 1D and 2D NMR data including HSQC and HMBC.     

Effect of polyamines on ribonuclease activity of rice (Oryza sativa L.)

The RNA content and RNase activity were determined during maturation and germination of rice seeds. The RNA content reached a maximum after the 16th day from anthesis but RNase activity steadily increased up to the last stage of maturation. During germination RNA content was greatest after 24 hr and associated with a very low level of RNase activity. Maximum RNase activity was observed at 72 hr from germination and it afterwards gradually declined. During germination, exogenous application of polyamines decreased the level of RNase activity. RNase was partially purified (448-fold) from 72 hr germinated embryonic axis. Effects of polyamines and other divalent cations were observed on the purified enzyme.