A colorimetric hydroxy naphthol blue based loop-mediated isothermal amplification detection assay targeting the ß-tubulin locus of Sarocladium oryzae infecting rice seed.

Sarocladium oryzae is a widely prevalent seed-borne pathogen of rice. The development of a rapid and on-site detection method for S. oryzae is therefore important to ensure the health of rice seeds. Loop-mediated isothermal amplification (LAMP) is ideal for field-level diagnosis since it offers quick, high-specific amplification of target template sequences at a single temperature. We designed primers based on the ß-tubulin region of S. oryzae. The LAMP technique devised was extremely sensitive, detecting the presence of the S. oryzae template at concentrations as low as 10 fg in 30 minutes at 65°C. The assay specificity was confirmed by performing the experiment with genomic DNA isolated from 22 different phytopathogens. Through the addition of hydroxy naphthol blue in the reaction process prior to amplification, a colour shift from violet to deep sky blue was seen in the vicinity of the target pathogen only. Finally, the LAMP assay was validated using live infected tissues, weeds and different varieties of seeds collected from different locations in Tamil Nadu, India. If developed into a detection kit, the LAMP assay developed in this study has potential applications in seed health laboratories, plant quarantine stations, and on-site diagnosis of S. oryzae in seeds and plants.

Untargeted Metabolite Profiling of Specialty Rice Grains Using Gas Chromatography Mass Spectrometry.

With-ever increasing demand food grains for the increasing population, it has also increased the importance of quality rice with nutritional and therapeutic properties. The quality of rice includes nutritional value, therapeutic properties, and further generation of aroma. Initial studies on sensory analysis using potassium hydroxide (1.7% KOH) identified the presence of a distinct aroma of the traditional rice cultivar Chakhao Amubi in comparison with other aromatic rice varieties were conducted. The metabolomic profiling of aromatic rice grains Chakhao Amubi, Pusa Basmati 1, and nonaromatic rice, Improved White Ponni was attempted to use gas chromatography-mass spectrometry (GC-MS). A total of fifty volatile aromatic compounds, including aromatic hydrocarbons, alkanes, alkenes, ketones, and aromatic aldehydes, have been identified. Detected compounds include six crucial volatile i.e., pentanal, hexanal, 2-pentylfuran, pyridine, (Z)-7-Decenal, and Mesitylene for distinct flavor and presence of aroma in Chakhao Amubi. The findings showed a distinct difference in the metabolic profile of Chakhao Amubi compared to Pusa Basmati 1 and Improved White Ponni. Thus, this study paved the way for a new understanding of the aromatic aspects of traditional rice germplasm and its utilization in rice breeding programs to improve the aroma, therapeutic, and nutritional characteristics of rice.

Screening tolerance to phosphorus starvation and haplotype analysis using phosphorus uptake 1 (Pup1) QTL linked markers in rice genotypes.

Phosphorus (P) deficiency tolerance is a pivotal trait for plant growth and development. Most of the commercial modern cultivars lack this trait and reported it as a very serious problem limiting crop productivity. This trait is advantageous if present in modern high yielding varieties as it increases the yield under the phosphorus-deficient soil conditions. With the importance of phosphorus deficiency tolerance, the present investigation was carried out with an objective to screen for tolerance to phosphorus deficiency using solution culture and phosphorus uptake 1 (Pup1) locus linked markers in 30 diverse rice genotypes. A wide range of varied responses to P deficiency in rice genotypes for all the traits were observed. Root length and enzyme activity showed increased mean performance under the - P condition when compared to + P condition. Medium to high heritability estimates were obtained for most of the traits. Correlation analysis showed that the traits: root P content, fresh shoot weight, dry shoot weight, and shoot length showed highly significant correlations with each other under - P conditions. Based on the hydroponics and molecular screening, three genotypes viz., ADT (R) 48, Improved Pusa Basmati 1 and UPLRI 5 were classified as tolerant for its response to P deficiency as they possessed significant increase in desirable root and shoot traits, increased acid phosphatase enzyme and these genotypes also possessed the Pup1 allele for all the five markers. The selected genotypes may be useful for the exploration of novel genes conferring phosphorus deficiency tolerance and used as donor parents in the breeding programs. Absence of this allele in the rice genotypes viz., drought tolerant (Anna (R) 4) and submergence tolerant (CR 1009 Sub 1) may warrant the development of multiple abiotic stress tolerance cultivars for upland and submergence cropping systems in future rice breeding program.

Structural and functional analyses of the wheat genomes based on expressed sequence tags (ESTs) related to abiotic stresses.

To gain insights into the structure and function of the wheat (Triticum aestivum L.) genomes, we identified 278 ESTs related to abiotic stress (cold, heat, drought, salinity, and aluminum) from 7671 ESTs previously mapped to wheat chromosomes. Of the 278 abiotic stress related ESTs, 259 (811 loci) were assigned to chromosome deletion bins and analyzed for their distribution pattern among the 7 homoeologous chromosome groups. Distribution of abiotic stress related EST loci were not uniform throughout the different regions of the chromosomes of the 3 wheat genomes. Both the short and long arms of group 4 chromosomes showed a higher number of loci in their distal regions compared with proximal regions. Of the 811 loci, the number of mapped loci on the A, B, and D genomes were 258, 281, and 272, respectively. The highest number of abiotic stress related loci were found in homoeologous chromosome group 2 (142 loci) and the lowest number were found in group 6 (94 loci). When considering the genome-specific ESTs, the B genome showed the highest number of unique ESTs (7 loci), while none were found in the D genome. Similarly, considering homoeologous group-specific ESTs, group 2 showed the highest number with 16 unique ESTs (58 loci), followed by group 4 with 9 unique ESTs (33 loci). Many of the classified proteins fell into the biological process categories associated with metabolism, cell growth, and cell maintenance. Most of the mapped ESTs fell into the category of enzyme activity (28%), followed by binding activity (27%). Enzymes related to abiotic stress such as beta-galactosidase, peroxidase, glutathione reductase, and trehalose-6-phosphate synthase were identified. The comparison of stress-responsive ESTs with genomic sequences of rice (Oryza sativa L.) chromosomes revealed the complexities of colinearity. This bin map provides insight into the structural and functional details of wheat genomic regions in relation to abiotic stress.

Analysis of expressed sequence tag loci on wheat chromosome group 4.

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.

Candidate defense genes from rice, barley, and maize and their association with qualitative and quantitative resistance in rice.

Candidate genes involved in both recognition (resistance gene analogs [RGAs]) and general plant defense (putative defense response [DR]) were used as molecular markers to test for association with resistance in rice to blast, bacterial blight (BB), sheath blight, and brown plant-hopper (BPH). The 118 marker loci were either polymerase chain reaction-based RGA markers or restriction fragment length polymorphism (RFLP) markers that included RGAs or putative DR genes from rice, barley, and maize. The markers were placed on an existing RFLP map generated from a mapping population of 116 doubled haploid (DH) lines derived from a cross between an improved indica rice cultivar, IR64, and a traditional japonica cultivar, Azucena. Most of the RGAs and DR genes detected a single locus with variable copy number and mapped on different chromosomes. Clusters of RGAs were observed, most notably on chromosome 11 where many known blast and BB resistance genes and quantitative trait loci (QTL) for blast, BB, sheath blight, and BPH were located. Major resistance genes and QTL for blast and BB resistance located on different chromosomes were associated with several candidate genes. Six putative QTL for BB were located on chromosomes 2, 3, 5, 7, and 8 and nine QTL for BPH resistance were located to chromosomes 3, 4, 6, 11, and 12. The alleles of QTL for BPH resistance were mostly from IR64 and each explained between 11.3 and 20.6% of the phenotypic variance. The alleles for BB resistance were only from the Azucena parent and each explained at least 8.4% of the variation. Several candidate RGA and DR gene markers were associated with QTL from the pathogens and pest. Several RGAs were mapped to BB QTL. Dihydrofolate reductase thymidylate synthase co-localized with two BPH QTL associated with plant response to feeding and also to blast QTL. Blast QTL also were associated with aldose reductase, oxalate oxidase, JAMyb (a jasmonic acid-induced Myb transcription factor), and peroxidase markers. The frame map provides reference points to select candidate genes for cosegregation analysis using other mapping populations, isogenic lines, and mutants.

The in vitro cytotoxicity of ascorbate depends on the culture medium used to perform the assay and involves hydrogen peroxide.

Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H2O2, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 +/- 39 microM H2O2 in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 +/- 17 microM H2O2 is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H2O2 at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H2O2 at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.

Hydrogen peroxide. Ubiquitous in cell culture and in vivo?

Hydrogen peroxide (H2O2) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H2O2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H2O2 may be greater than is commonly supposed; levels of H2O2 in the human body may be controlled not only by catabolism but also by excretion, and H2O2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Cell culture is a widely used method for the investigation of "physiological" processes such as signal transduction and regulation of gene expression, but chemical reactions involving cell culture media are rarely considered. Addition of reducing agents to commonly used cell-culture media can lead to generation of substantial amounts of H2O2. Some or all of the reported effects of ascorbic acid and polyphenolic compounds (e.g., quercetin, catechin, epigallocatechin, epigallocatechin gallate) on cells in culture may be due to H2O2 generation by interaction of these compounds with cell culture media.

Superoxide anion inhibits drug-induced tumor cell death.

Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.