Development and application of two novel functional molecular markers of BADH2 in rice

BACKGROUND: Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. RESULTS: In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2- E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F2 population. CONCLUSIONS: Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.

Possible Metsulfuron Herbicide Detoxification by a Oryza sativa L. Glutathione S-transferase Enzyme

Abstract Rice (Oryza sativa L.) is one of the most important crops in the world, and it is considered the primary source of nutritional layout in developing countries in Asia. The glutathione S-transferases (GSTs) superfamily confers to rice protection against biotic and abiotic stress, and herbicide resistance. However, the three-dimensional structure of a GST Tau class, is unsolved. The objectives of this work were to develop a reliable comparative model for the s-transferase glutathione class Tau 4 from rice, and simulate docking interactions, against herbicides bentazon and metsulfuron. Results showed that the predicted model is reliable and has structural quality. Ramachandran plot set 91.9% of the residues in the most favored regions. All complexes showed negative binding energies values; and metsulfuron docked to the glutathione tripeptide, and it represents a possible insilico evidence of glutathione conjugation with this herbicide.

Dark septate endophyte decreases stress on rice plants

Abstract Abiotic stress is one of the major limiting factors for plant development and productivity, which makes it important to identify microorganisms capable of increasing plant tolerance to stress. Dark septate endophytes can be symbionts of plants. In the present study, we evaluated the ability of dark septate endophytes isolates to reduce the effects of water stress in the rice varieties Nipponbare and Piauí. The experiments were performed under gnotobiotic conditions, and the water stress was induced with PEG. Four dark septate endophytes were isolated from the roots of wild rice (Oryza glumaepatula) collected from the Brazilian Amazon. Plant height as well as shoot and root fresh and dry matter were measured. Leaf protein concentrations and antioxidant enzyme activity were also estimated. The dark septate endophytes were grown in vitro in Petri dishes containing culture medium; they exhibited different levels of tolerance to salinity and water stress. The two rice varieties tested responded differently to inoculation with dark septate endophytes. Endophytes promoted rice plant growth both in the presence and in the absence of a water deficit. Decreased oxidative stress in plants in response to inoculation was observed in nearly all inoculated treatments, as indicated by the decrease in antioxidant enzyme activity. Dark septate endophytes fungi were shown to increase the tolerance of rice plants to stress caused by water deficiency.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

Bioethanol production from rice straw residues

A rice straw -cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 ºC, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L-¹.

Comparison of microsatellites and isozymes in genetic diversity studies of Oryza glumaepatula (Poaceae) populations

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A P=3.17, Ho=0.081, He=0.351) than isozyme loci (A=1.20, p=0.20, A P=1.38, Ho=0.006, He=0.056). Interpopulation genetic differentiation detected by SSR loci (R ST=0.631, equivalent to F ST=0.533) was lower than that obtained with isozymes (F ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ( =0.14) was higher than that obtained using isozymes ( =0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined ...

El estudio de la estructura genética de poblaciones de plantas silvestres es esencial para su manejo y conservación. Varios marcadores de ADN e isoenzimas se han utilizado en este tipo de análisis. Con el fin de proporcionar una mejor comprensión de los resultados obtenidos y saber que marcador codominante elegir para futuros estudios en poblaciones naturales de Oryza glumaepatula, este trabajo busco evaluar y comparar dos marcadores de ADN, isoenzimas y microsatélites, en la diversidad y estructura genética de 13 poblaciones, destacando las similitudes y divergencias de cada marcador, así como la importancia relativa de los resultados en genética de poblaciones y conservación. Para los SSR, ocho loci SSR fueron evaluados, y los fragmentos se visualizaron utilizando el procedimiento de coloración con plata. Los análisis de isoenzimas se realizaron en geles de poliacrilamida, en los seis loci enzimáticos. Los loci SSR mostraron mayores niveles de diversidad genética que los loci isoenzimáticos, en promedio. La diferenciación genética entre los loci SSR (R ST=0.631, equivalente a F ST=0.533) fue inferior a la obtenida con las isoenzimas (F ST=0.772). Ambos marcadores mostraron alta desviación del equilibrio de Hardy-Weinberg (F IS=0.744 y 0.899, respectivamente, para SSR e isoenzimas). La tasa media aparente de cruzamiento para SSR ( =0.14) fue mayor que la obtenida con isoenzimas ( =0.043), aunque ambos marcadores detectaron niveles más bajos en la tasa de fecundación cruzada para la Amazonia, en comparación con la región del Pantanal. La estimación de número de migrantes también fue mayor para los SSR (Nm=0.219) que en isoenzimas (Nm=0.074). No se obtuvo ninguna correlación entre las distancias genéticas y geográficas para los SSR, y para las isoenzimas se obtuvo una correlación positiva entre las distancias genéticas y geográficas. Llegamos a la conclusión de que estos marcadores son divergentes en la detección de los parámetros de la diversidad genética en O. glumaepatula y que los microsatélites son más eficientes para detectar la información a nivel intra-poblacional, mientras que las isoenzimas son más potentes para detectar la diversidad entre poblaciones.

Physiological studies on carboxymethyl cellulase formation by Aspergillus terreus DSM 826

Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30ºC for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.

Expression of rna polymerase IV and V in oryza sativa

RNA polymerase IV and V are principal players in the RdDM pathway, where their current study has shown interaction of several factors that control DNA silencing of intergenic regions and siRNA production. DNA silencing is an important process during cell differentiation, nuclear structure and viral control. However, RNA pol IV and V are yet to be study in model monocot systems like Oryza sativa that can provide further information on genetic silence mechanism in plats. We show the expression pattern of these polymerases in tissues extracts of Oryza sativa. Detectable amounts of these polymerases are found in specific adult plant tissues and particularly expressed during somatic embryogenesis but not during early stages of normal embryo development. The use of synthetic auxin leads to an induction of both RNA pol IV and V in scutellum tissue where nuclear localization may be required for genome reorganization and gene silencing.

Isolation of stress responsive Psb A gene from rice (Oryza sativa l.) using differential display.

Differential display (DD) experiments were performed on drought-tolerant rice (Oryza sativa L.) genotype N22 to identify both upregulated and downregulated partial cDNAs with respect to moisture stress. DNA polymorphism was detected between drought-stressed and control leaf tissues on the DD gels. A partial cDNA showing differential expression, with respect to moisture stress was isolated from the gel. Northern blotting analysis was performed using this cDNA as a probe and it was observed that mRNA corresponding to this transcript was accumulated to high level in rice leaves under water deficit stress. At the DNA sequence level, the partial cDNA showed homology with psb A gene encoding for Dl protein.

Effects of light on the feedback control of GA-20 oxidase gene homolog in DongJinByeo seedlings.

The effects of gibberellin (GA) on the expression of GA-20 oxidase gene homolog were examined in light-grown seedlings and dark-grown seedlings of DongJinByeo. The growth rates of the stems of etiolated seedlings were faster than those of green seedlings. However, upon addition of GA to these seedlings, the stem growth rates of green seedlings were faster than those of etiolated seedlings. To understand the molecular mechanism of GA gene regulation in DongJinByeo, total RNA from DongJinByeo was hybridized with cDNA of GA-20 oxidase gene homolog. Greater accumulation of transcript of GA-20 oxidase gene homolog was observed in green seedlings than in etiolated seedlings. However, upon addition of GA, higher accumulation of the gene transcript was found in etiolated seedlings than in green seedlings, indicating that expression of the transcript of GA-20 oxidase gene homolog might be inhibited by light. These results suggest that light might regulate feedback control of the transcript of GA-20 oxidase gene homolog in DongJinByeo.

Expression of delta1-pyrroline-5-carboxylate synthetase gene during drought in rice (Oryza sativa L.).

The 45-days-old seedlings of drought resistant (N-22, CR143-2-2) and susceptible rice (Oryza sativa L.) genotypes (Panidhan, Pusa-169) were subjected to osmotic stress in PEG-6000 solution of -10 and -16 bar and the relative water content (RWC), proline content, and pyrroline-5-carboxylate synthetase (P5CS) activity and its P5CS expression were studied. A gradual decrease in RWC was observed in tolerant genotypes, whereas the decrease was drastic in susceptible ones. Proline content and P5CS activity increased both in susceptible and tolerant genotypes; the increase was higher in tolerant genotypes. Higher proline levels in tolerant genotypes were due to increased P5CS activity. The EcoRI, BamHI and XbaI restricted DNA of N-22 and Panidhan genotypes were hybridized with Arabidopsis P5CS sequence and a single band (approx 2.4 kb) was observed, however, P5CS expression was more in N-22 as compared to Panidhan.

A partial sequence of lipoxygenase gene from genomic DNA of aromatic rice (Oryza sativa L.).

Aromatic (Bas-370, PB-1) and non-aromatic (Pusa-677, Pusa-834) rice were selected for the characterization and for distribution of lipoxygenase (Lox) genes. Polymorphism was observed when genomic DNA of rice varieties was hybridized with a heterologous lipoxygenase probe. A distinct polymorphic fragment (approximately 1.2 kb) was found in Bas-370. Sub-genomic library of Bas-370 was constructed and screened with LoxA probe. The smallest putative clone (pBas-14) of approximately 1.2 kb was sequenced. Complete nucleotide and deduced amino acid sequence showed the clone was 1134 bp long and comprised of 378 amino acid residues. PCR amplification of genomic DNA from four rice varieties with a soybean Lox primer also showed a polymorphic fragment of size approximately 600 bp (amplicon) in aromatic varieties that was sequenced directly. Nucleotide sequence alignment between pBas-14 and amplicon concluded that the amplicon was a part of the insert pBas-14.

Environmental stress-induced extracellular isoperoxidase RC3 from rice.

Effects of various environmental stresses such as heavy metals, salts and low (high) temperature on the secretion of peroxidase isozyme into the medium were examined in rice (Oryza sativa cv. Nak-Dong) suspension culture. The major extracellular peroxidases secreted into the medium by various stresses were cationic isoperoxidases. A far migrating cationic isoperoxidase RC3 was isolated from the medium after application of CaCl2, the effective stimulator for peroxidase secretion. Isolation of extracellular isoperoxidase RC3 was accomplished by ammonium sulfate fractionation, CM-cellulose cation-exchange chromatography, and Sephacryl S-100 gel filtration. The enzyme was a glycoprotein having molecular weight of approximately 34 KDa as determined by SDS-PAGE and 38 KDa by Sephacryl S-100 gel filtration. The pI value of the enzyme was 8.9. Kinetic studies revealed that the optimum pH of the enzyme was 6.0 for guaiacol and H2O2, and the Km values for guaiacol and H2O2 were 10.5 mM and 3.2 mM, respectively.

Bio-indicators of soil fertility in rice rotation

The relationship between enzyme activities and some soil properties under rice rotation at two governorates was studied to find out whether or not the enzyme activities can be taken as a bioindicator to soil fertility. The results indicated that there are clear differences in soil enzyme activities [dehydrogenase, phosphatase, invertase and urease] at the different localities of the two governorates according to its soil fertility level. Significant positive correlation was established between the activities of dehydrogenase and urease and total bacterial counts, total fungi, phosphate-dissolving bacteria [PDB], ureolytic microorganisms and CO2 evolution. Phosphatase activity was positively correlated with total baccarat counts, total fungi and CO2 evolution but not correlated with PDB and ureolytic organisms. On the other hand, positive correlations were detected between invertase activity and each of total fungi, PDB, ureolytic organisms and evolved CO2

Modification of the redox state of cytochrome c oxidase of rice due to certain stress treatments.

The redox state of cytochrome alpha 3 during in situ respiration of leaves of 20-day-old rice seedlings was assessed by in vivo aerobic assay of nitrate reductase, after 1 min exposure to carbon monoxide. Different stress treatments like water and salt stresses, disintegration of leaf tissues and darkness modified the redox state of cytochrome c oxidase. The dark treatment altered the redox state of cytochrome oxidase from reduced to the oxidized state, as judged by its reaction with CO in CO-sensitive rice cultivar. The water and salt stresses as well as the disintegration of leaf tissue on the contrary altered cytochrome oxidase from the oxidized to its reduced state in CO-insensitive cultivars; probably by changing the cellular integrity, turgidity and structure of mitochondrial membrane, and also due to decreased mitochondrial energization.