Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 μm and 50 μm As₂O₃) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Genetic analysis of endosperm mutants in rice Oryza sativa L.

Summary Sugary, shrunken, floury, white core, amylose extender and dull mutants induced in japonica varieties were used in this study. The results of an allelic analysis conducted in japonica background indicated that the two sugary mutants 82GF and EM5 are allelic. The two amylose extender mutants 2064 and EM16 are also allelic. The opaque mutant ESD7-3(0) and floury mutants 2047, EM17 and EM28 are allelic as well and have the flo-1 gene. The three white core mutants EM3, EM24 and EM66 were found to be non-allelic. Eleven dull mutants were investigated. Dull mutants 2057, 2083, 2091 and EM15 were found to be allelic to each other. Similarly, dull mutants 2077, 2078 and 2120 have allelic genes. Dull mutants 2035, EM12, EM47, and EM98 are non-allelic to the above loci. Dull genes in EM12, EM15, and EM98 were designated earlier as du-1, du-2 and du-4, respectively.The mutant genes were transferred to indica background by two backcrosses to IR36. Some of the mutant genes were located to respective chromosomes through trisomic analysis using primary trisomics of IR36. In this way the amylose extender gene ae was located to chromosome 2, the flo-1 was located to chromosome 5 and the flo-2 to chromosome 4. Dull genes of EM47, 2120, and 2035 were assigned to chromosomes 6, 9, and 6, respectively.     

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 μM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 – 120 h, an increased level of protein as well as free amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50–100 μM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 μM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression of protease and peptidase activities due to Cd treatments in germinating rice seeds leading to altered levels of protein and amino acids.     

Inhibition of ribonuclease and protease activities in germinating rice seeds exposed to nickel

When the seeds of two rice cvs. Malviya-36 and Pant-12 were germinated up to 120 h in the presence of 200 and 400 μM NiSO₄, a significant reduction in the germination of seeds occurred. Seeds germinating in the presence of 400 μM NiSO₄ showed about 12–20% decline in germination percent, about 20–53% decline in lengths and about 8–34% decline in fresh weights of roots and shoots at 120 h of germination. Ni²⁺ exposure of germinating seeds resulted in apparent increased levels of RNA, soluble proteins, and free amino acids in endosperms as well as embryo axes. A 400 μM Ni²⁺ treatment led to about 58–101% increase in the level of soluble proteins and about 39–107% increase in the level of free amino acids in embryo axes at 96 h of germination. Activities of ribonuclease and protease declined significantly with increasing levels of Ni²⁺ treatment. Isoenzyme profile of RNase as revealed by activity staining indicated decline in the intensities of 3–4 preexisting enzyme isoforms in embryo axes of both the rice cultivars and disappearance of one of the two isoforms in endosperms of cv. Pant-12 due to 400 μM Ni²⁺ treatment. Results suggest that the presence of high level of Ni²⁺ in the medium of germinating rice seeds serves as a stress factor resulting in decreased hydrolysis as well as delayed mobilization of endospermic RNA and protein reserves and causing imbalance in the level of biomolecules like RNA, proteins, and amino acids in growing embryo axes. These events would ultimately contribute to decreased germination of rice seeds in high Ni²⁺ containing environment.     

Effect of jasmonic acid on endogenous gibberellins and abscisic acid in rice under NaCl stress

Content of endogenous abscisic acid (ABA) increased in rice plants under salt stress. Pre- or post-treatment by jasmonic acid (JA) mostly further increased ABA content. In the presence of salt stress also content of gibberellins (GAs) mostly increased more after treatment by JA. Endogenous content of bioactive GA₁ was higher in post-treatment by JA than in pre-treatment by JA.This study was supported by the Korea Science and Engineering Foundation (2000-2-20100-001-3)     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

The importance of ethanolic fermentation for primary root growth of germinating rice under anoxia

The relationship between primary root growth and ethanolic fermentationwas investigated in five cultivars of germinating rice (Oryza sativa L.) seeds subjected to 48 h-anoxic stress. The anoxic stressinhibited the growth of all rice cultivars, however, there were significantdifferences in the growth among the cultivars. The stress increased alcoholdehydrogenase activities and ethanol concentrations in the roots of all ricecultivars but there were differences between cultivars in the activity and theconcentration. The ethanol concentrations in the roots were closely correlatedwith the root growth of the corresponding cultivars. These results suggest thatability to induce ethanolic fermentation may play an important role in theprimary root growth of germinating rice in anoxic condition.     

Molecular Cloning and Expression of a cDNA Encoding Lon Protease from rice (Oryza sativa)

The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein.     

Effects of 6-methoxy-2-benzoxazolinone on the germination and α-amylase activity in lettuce seeds

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of α-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration–response curves for the germination and α-amylase indicate that the percentage of the germination was positively correlated with the activity of α-amylase in the seeds. Lettuce seeds germinated around 18 h after incubation and inhibition of α-amylase by MBOA occurred within 6 h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of α-amylase activity.     

Hormones in the grains in relation to sink strength and postanthesis development of spikelets in rice

Inferior spikelets usually exhibit a slower grain filling rate and lower grain weight than superior spikelets in a rice (Oryza sativa L.) panicle. This study investigated whether the variations in grain filling between the two kinds of spikelets were attributed to their sink strength and whether the sink strength was regulated by the hormonal levels in the grains. Using two field-grown rice genotypes, the division rate of endosperm cells, hormonal levels in the grains, and grain weight of both superior and inferior spikelets were determined during the grain filling period. The results showed that superior spikelets had dominance over inferior spikelets in endosperm cell division rate and cell number, grain filling and grain weight. Changes in zeatin (Z) and zeatin riboside (ZR) contents paralleled and were very significantly correlated with the cell division rate and cell number. Cell division rate and the content of indole-3-acetic acid (IAA) in the grains were also significantly correlated. Gibberellin (GAs; GA₁+ GA₄) content of the grains was high but ABA levels were low at the early grain filling stage. ABA increased substantially during the linear phase of grain growth and was very significantly correlated with grain dry weight during this period. Application of kinetin at 2 through 6 days post anthesis (DPA) significantly increased cell number, while spraying ABA at 11 through 15 DPA significantly increased the grain filling rate. The results suggest that differences in sink strength are responsible for variations in grain filling between superior and inferior spikelets. Both cytokinins and IAA in the grains may mediate cell division in rice endosperm at early grain filling stages, and therefore regulate the sink size of the grain, whereas ABA content correlates with sink activity during the linear period of grain growth.     

Influence of boric acid on somatic embryogenesis of a cytosterile line of indica rice

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22 μM to the embryo induction medium containing basal salts, with 9.84 μM 2,4-dichlorophenoxyacetic acid. Boric acid was found to exert different effects on scutellum, coleoptile and root tissues. Increased growth of embryogenic masses with increasing boric acid concentrations was observed on coleoptile tissues of mature seed. Embryogenic response was observed at 161.29 μM boric acid on coleoptile and 241.93 μM boric acid on root tissue. Scutellum tissue showed embryonic response in 161.29 and 241.93 μM boric acid. The results indicate that the boric acid used in standard Murashige and Skoog medium is not sufficient to support induction of somatic embryogenesis in IR-58025 A. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa andO. glaberrima) contain more than 18% amylose in endosperm starch, whilewaxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even inwaxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with aMr of 60 kd, whereaswaxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is thewaxy protein encoded by theWx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands withMr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum againstWx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous toWx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from theWx gene expressed in the endosperm.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Effect of End-of-day far-red light exposures on fertility alteration and flowering in photoperiod-sensitive genic male-sterile rice

The rice photoperiod-sensitive genic male-sterile mutant (PGMR) is sterile under long days, but fertile in short days. Phytochrome is involved in the photoperiod-induced male-sterile process. To investigate the mechanisms, of phytochrome action in PGMR, end-of-day (EOD) experiments were carried out. Flowering in PGMR was delayed considerably by EOD far-red light exposures following a short day of 10 hr, whereas its fertility decreased to the same extent as the original line. This result suggests that photoperiod response mediating fertility alteration in PGMR somewhat differed from that in flowering,i.e., fertility alteration and flowering might be under the separate phytochrome signaling control.     

Effect of polyamines and guanidines on the growth,nitrogen assimilation and reserve mobilization in germinating radish seeds

Polyamines and guanidines enhanced the growth of radish seedlings grown in dark or light, irrespective of the supply of nitrogen. All the compounds inhibited ntirate reducatase and glutamine synthetase in the cotyledons of light-grown but not in dark-grown seeds. Nitrite reductase and glutamate dehydrogenase were not affected. Protease activity was enhanced by all the compounds in dark-as well as in light-grown seeds. Alanine aminotransferase activity was increased only in the light-grown seeds. The inhibition of nitrate reductase was not due to decreased nitrate uptake but was due to a decreased metabolic pool of nitrate and a decline in enzyme synthesis. The inhibition of glutamine synthetase and activation of alanine aminotransferase by the compounds was found only in the chloroplast fraction. The activation of protease was due to the release or activation of preexisting enzyme while that of alanine aminotransferase was dependent on the de novo protein synthesis which was abolished by cycloheximide.     

Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.