Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.

Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid.

The rice OsLpa1 gene encodes a novel protein involved in phytic acid metabolism.

The rice low phytic acid 1 (lpa1) mutant was originally identified using a forward genetics approach. This mutant exhibits a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, it does not appear to differ significantly in productivity from its wild-type progenitor. A second lpa1 mutant was identified from additional screening for high seed inorganic phosphorus phenotypes. Using a positional cloning strategy, we identified a single candidate gene at the rice Lpa1 locus. Sequence analysis of the candidate gene from the lpa1 mutants revealed two independent mutations (a single base pair substitution and a single base pair deletion) that confirmed the identification of this candidate as the rice low phytic acid 1 gene, OsLpa1. The OsLpa1 gene has three splice variants. The location and nature of the two mutations suggests that these lesions only affect the translation of the predicted protein derived from the longest transcript. The proteins encoded by OsLpa1 do not have homology to any of the inositol phosphate metabolism genes recently characterized in plants, although there is homology to 2-phosphoglycerate kinase, an enzyme found in hyperthermophilic methanogens that catalyzes the formation of 2,3-bisphosphoglycerate from 2-phosphoglycerate. OsLpa1 represents a novel gene involved in phytic acid metabolism.

Determination of ammonium ion by fluorometry or spectrophotometry after on-line derivatization with o-phthalaldehyde.

A fast, sensitive, simple, and highly reproducible method for routine assay of ammonium ion (NH4+) was developed by using HPLC equipment. The method is based on the reaction of NH4+ with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol. After an on-line derivatization, the resulting NH4(+)-OPA product was quantified by using fluorometric or spectrophotometric detection. For fluorometric detection, the excitation and emission wavelengths were 410 and 470 nm, respectively. The spectrophotometric detection was made by measuring absorbance at 410 nm. Results on the effects of OPA-reagent composition and pH, reaction temperature, sample matrix, and linearity of the assay are presented. Even though it took about 2 min from the time of sample injection to the appearance of sample peak, sample injections could be overlapped at an interval of about 1 min. Thus, the actual time needed for analysis was about 1 min per assay. The method can be used in a fully automated mode by using an autosampler injector.

The uptake of NO3-, NO2-, and NH4+ by intact wheat (Triticum aestivum) seedlings. I. Induction and kinetics of transport systems.

The inducibility and kinetics of the NO3-, NO2-, and NH4+ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO3- and NO2- transporters. Evidence for substrate inducibility of the NH4+ transporters requiring 5 hours is presented. The transport of NO3- was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO2- and NH4+. The Km values for NO3-, NO2-, and NH4+ were, respectively, 0.027, 0.054, and 0.05 millimolar. The Km for mechanism II of NO3- transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.

Potentiometric studies of the complexes of chromium(VI), molybdenum(VI) and tungsten(VI) with some Azoxine S dyes.

The equilibrium constants of the complexation reactions of Cr(VI), Mo(VI) and W(VI) with 8-hydroxyquinoline-5-sulphonic acid (OXS), 7-phenylazo-8-hydroxyquinoline-5-sulphonic acid (PAZOXS), 7-(4-sulphophenylazo)-8-hydroxyquinoline-5-sulphonic acid (SPAZOXS) and 7-(4-sulphonaphthylazo)-8-hydroxyquinoline-5-sulphonic acid (SNAZOXS) have been determined by potentiometric pH titration. The values in the case of chromate are different from those for molybdate and tungstate. The order of stabilities is OXS > PAZOXS > SPAZOXS > SNAZOXS.

Spectrophotometric determination of zirconium with 2-(1-hydroxy-4,6-dinitro-2-phenylazo)-1,8-dihydroxynaphthalene-3,6-disulphonate (picramine ca) as chromogenic reagent.

The title compound has been used as a selective reagent for the micro-determination of zirconium in acidic medium (0.5MHCl) and found to be better than Picramine R. Spectrophotometric studies show the formation of a 2:1 (ligand:Zr) water-soluble complex and the reaction is suitable for photometric determination of 0.4-2.8 ppm of zirconium. The colour takes about 90 min to develop fully and is stable for about 20 hr. The molar absorptivity of the complex is 2.4 x 10(4) and the equilibrium constant is of the order of 10(10). The interference due to a number of ions has been studied.

Studies on vanadium(IV) complexes of azoxine s dyes.

Comparative studies of the reaction of 7-arylazo-8-hydroxyquinoline-5-sulphonic acid (Azoxine S) dyes with vanadium(IV), show that 2:1 yellow, water-soluble complexes are formed over the pH range 2.5-6, and that the phenyl derivative is the most suitable for spectrophotometric determination of 0.2-1.4 ppm of vanadium(IV). The colour is formed instantaneously and is stable for about 8 hr. The molar absorptivity at lambda(max), 400 mmu;, is 1.15 x 10(4), and the equilibrium constant for complex formation is of the order of 10(2). These dyes can also be used as indicators in the direct complexometric determination of vanadium(IV). The interference of a number of anions and cations is reported.