Sorghum Leaf Research Articles

Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase

The photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca2+-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [32P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C4-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca2+-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK.

Sensitivities of Stress Indicators for Available Soil Water and Its Salinity and for Sorghum Leaf Water Potential under Drip Irrigation

The impact of two input amounts, two frequencies, and three salinity levels on the temporal sensitivity of the stress indicators, available soil water (K W ), its salinity (K S ), and leaf water potential of sorghum [Sorghum bicolor (L.)] grown in dune sand in a plastic greenhouse were investigated. The K W for the irrigation input amount equivalent to one‐half of an open pan on a daily basis (EP0.50‐1) ranged from 0.32 to 0.51 compared with 0.51 to 0.98 for the 100% of open‐pan irrigation input on daily basis. The corresponding K S ranges were 0.50 to 0.98 (EP1.00‐1) and 80 to 0.98 for K s . The K W for the three increasing salinity levels (S‐1, S‐2, and S‐3) ranged from 0.68 to 0.96 for S‐1, 0.58 to 0.90 for S‐2, and 0.43 to 0.89 for S‐3, respectively, and from 0.85 to 1.00 for the control. The corresponding K S ranges were 0.89 to 0.98, 0.70 to 0.96, 0.49 to 0.70, and 1.00 to 1.00, respectively. The LWP ranges for the three salinity levels were −1.70 to −0.70 for S‐1, −2.50 to −1.20 for S‐2, −2.90 to −1.60 for S‐3, and −0.70 to −0.20 MPa for the control. The time series statistical parameters mean, median, coefficient of variation (CV), and the 20th percentile values (PC20), assessed to discriminate the temporal sensitivity of the stress indicators indicated that CV and PC20 are equally better than median, which in turn is better than mean. Sorghum grain yield depended on irrigation input amount and schedules, leaf water potential (LWP), and the interaction involving K W and K S (R2 adj 0.71 to 0.85). The temporal sensitivity of the stress indicators K W , K S , and LWP indicated that EP1.00‐1 provided the least‐risk irrigation option with regard to water and salinity stress in relation to sorghum grain yield.

Structure and Properties of Natural Cellulose Fibers Obtained from Sorghum Leaves and Stems

For the first time, sorghum leaves and stems have been used to produce natural cellulose fibers with properties suitable for composite, textile, and other high-value fibrous applications. The leaf and stems fibers produced are multicellular and have similar cellulose contents. The breaking tenacity and elongation of the fibers are similar to that of natural cellulose fibers such as kenaf and cornstalk fibers. However, the sorghum fibers have a modulus of about 113 g/denier (15 GPa) similar to the modulus of cornstalk fibers but higher than that of cotton and cornhusk fibers. At least 7 million tons of natural cellulose fibers can be produced by using the sorghum stems and leaves available as byproducts every year. Using the sorghum byproducts as a source for cellulose fibers will help to add value to the sorghum crops and also make the fiber industry more sustainable.

Distribution of sugarcane mosaic and sugarcane streak mosaic virus in India

Seven (sugarcane and sorghum) leaf samples exhibiting mosaic symptoms and that originated from (Uttar Pradesh, Maharashtra and Tamil Nadu) were characterized through RT-PCR assays with specific designed primers forSugarcane mosaic virus, SCMV (F4 and R3),Sugarcane streak mosaic virus, SCSMV(ST2 and P1) andSorghum mosaic virus, SrMV (F3 and R3). SCMV was detected by RT-PCR in 3 sugarcane varieties. The expected 0.9 kbp SCMV fragment was amplified by RT-PCR with the SCMV F4 and SCMV R3 primers with mosaic samples from Maharashtra (cv.VSI-9-20 and CoM 9086) and Tamil Nadu (cv. Co740). However, ca 0.5 kbp fragment was amplified from six of the samples out of seven tested using primers SCSMV-ST2 and SCSMV-P1. RT-PCR reactions were negative with SrMV primers pair in all the tested samples. Our results suggested that SCSMV is found more commonly associated with sugarcane mosaic samples in India in comparison to the SCMV. Interestingly, mixed infection of SCMV and SCSMV was also detected in sugarcane mosaic infected leaf samples from Maharashtra and Tamil Nadu.

Antibiosis mechanism of resistance to spotted stem borer, Chilo partellus in sorghum, Sorghum bioclor

Spotted stem borer, Chilo partellus (Swinhoe), is the most important pest of sorghum in Asia and South and Eastern Africa, and host plant resistance is an important component for controlling this pest under subsistence farming conditions. Therefore, we studied the antibiosis mechanism of resistance in a diverse array of 20 sorghum genotypes at the seedling stage by incorporating the freeze-dried leaf powder into artificial diet. Freeze-dried sorghum leaf powder at 12.5 g per 250 ml of the standard artificial diet or replacement of chickpea flour in the artificial diet by 50% with sorghum leaf powder can be used to quantify the extent of antibiosis mechanism of resistance to C. partellus in sorghum. There was a significant variation in larval survival, larval and pupal weights, larval and pupal periods, and percentage pupation and adult emergence in diets impregnated with freeze-dried leaf powder of different sorghum genotypes. Sorghum genotypes such as IS 1044, IS 2123, IS 1054, IS 18573, and ICSV 714 showed antibiosis to C. partellus in terms of reduced survival and development. Principal component analysis indicated that there is considerable diversity in sorghum genotypes for antibiosis to C. partellus. Genotypes placed in different groups can be used in resistance breeding programs to diversify the basis of resistance to this pest.

Nitrogen deficiency effects on plant growth, leaf photosynthesis, and hyperspectral reflectance properties of sorghum

An experiment was conducted under outdoor pot-culture conditions to determine effects of nitrogen (N) deficiency on sorghum growth, physiology, and leaf hyperspectral reflectance properties. Sorghum (cv. DK 44C) was seeded in 360 twelve-litre pots filled with fine sand. All pots were irrigated with half-strength Hoagland's nutrient solution from emergence to 25 days after sowing (DAS). Thereafter, pots were separated into three identical groups and the following treatments were initiated: (1) the control (100% N) continued receiving the half-strength nutrient solution; (2) reduced N to 20% of the control (20% N); and (3) withheld N from the solution (0% N). Photosynthetic rate (Pn), chlorophyll (Chl) and N concentrations, and hyperspectral reflectance of the uppermost, fully expanded leaves were determined at 3- to 4-day-interval from 21 to 58 DAS during the N treatments. Plants were harvested 58 DAS to determine effects of N deficiency on leaf area (LA), biomass accumulation, and partitioning. Nitrogen deficiency significantly reduced LA, leaf Chl content and Pn, resulting in lower biomass production. Decreased leaf Pn due to N deficiency was mainly associated with lower stomatal conductance rather than carboxylation capacity of leaf chemistry. Among plant components of dry weights, leaf dry weight had the greatest and root dry weight had the smallest decrease under N deficiency. Nitrogen-deficit stress mainly increased leaf reflectance at 555 ( R 555) and 715 nm ( R 715) and caused a red-edge shift to shorter wavelength. Leaf N and Chl concentrations were linearly correlated with not only the reflectance ratios of R 405/ R 715 ( r 2 = 0.68 ***) and R 1075/ R 735 ( r 2 = 0.64 ***), respectively, but also the first derivatives of the reflectance (d R/d λ) in red edge centered 730 or 740 nm ( r 2 = 0.73–0.82 ***). These specific reflectance ratios or d R/d λ may be used for rapid and non-destructive estimation of sorghum leaf Chl and plant N status.

Isolation and characterization of genomic and transcribed retrotransposon sequences from sorghum.

Reverse transcriptase sequences from both major classes of retrotransposons were amplified from sorghum genomic DNA, leaf mRNA and callus protoplast mRNA. Sequence analysis of clones derived from genomic DNA demonstrated the presence of a wide variety of copia-like and gypsy-like elements. Twenty-four families of copia-like elements were found, of which at least thirteen were expressed in callus protoplasts. Two families (containing forty-eight subfamilies) of gypsy-like elements were discovered, both closely related to Huck of maize. At least twenty-seven of these subfamilies were expressed in callus protoplasts. Most of these elements were expressed at high levels in protoplasts derived from embryogenic callus, but expression of only a few was detected (at low levels) in leaves. Sequence divergence within individual families was quite high, and all relatedness profiles were consistent with vertical transmission of these elements. These data indicate that sorghum contains a large number and diversity of retrotransposons, and that some may be useful as transposon tagging systems in callus protoplasts.

Raman spectroscopic analysis of cyanogenic glucosides in plants: development of a flow injection surface-enhanced Raman scatter (FI-SERS) method for determination of cyanide.

Cyanogenic glucosides were studied using Raman spectroscopy. Spectra of the crystal forms of linamarin, linustatin, neolinustatin, amygdalin, sambunigrin, and dhurrin were obtained using a Raman spectrograph microscope equipped with a 532 nm laser. The position of the signal from the C identical with N triple bond of the cyanohydrin group was influenced by the nature of the side group and was above 2240 cm(-1) for the three cyanogenic glucosides that contain a neighboring aromatic ring, and below or partially below 2240 cm(-1) for the non-aromatic cyanoglucosides. Signals from the CN bond of linamarin/lotaustralin in leaves and roots from a medium cyanogenic cassava variety were obtained in situ using a Fourier transform near-infrared (FT-NIR) Raman interferometer with a 1064 nm laser, but the signal was very weak and difficult to obtain. A spectrum containing a signal from the CN bond of dhurrin in a freeze-dried sorghum leaf was also obtained using this instrument. Surface-enhanced Raman Spectroscopy (SERS) was demonstrated to be a more sensitive method that enabled determination of the cyanogenic potential of plant tissue. The SERS method was optimized by flow injection (FI) using a colloidal gold dispersion as effluent. Potential problems and pitfalls of the method are discussed.

Localization of antioxidant enzymes in the cellular compartments of sorghum leaves

The localization of antioxidant enzymes between the mesophyll and bundle sheath cells were determined in sorghum (Sorghum vulgare L.) leaves. The activity of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) were assayed in whole leaf, mesophyll and bundle sheath fractions of sorghum leaves subjected to water-limited conditions. Drought was imposed by withholding water and the plants were maintained at different water potentials ranging from 0.5–2.0 MPa. The purity of the isolates was tested using the marker enzymes like RuBPcase and PEPcase. GR was mostly localized in mesophyll fraction, while SOD, APX and peroxidase were located in bundle sheath cells. Catalase was found to be equally distributed between the two cell types. Under water stress conditions, most of the SOD activity was found in the bundle sheath tissues. Little or no activity of the enzymes CAT, APX or POD was found in the mesophyll extracts when exposed to water stress. GR activity increased when exposed to low water regimes. From this study, it is clear that antioxidants are differentially distributed between the mesophyll and bundle sheath cells in sorghum leaves. Under water stress conditions, the mesophyll cells showed less damage from oxidative stress when compared to the bundle sheath cells. This is critical for determining the sensitivity of sorghum to extreme climatic conditions.

MECHANISM OF SALT TOLERANCE OF TWO OLIVE OLEA EUROPAEA L. CULTIVARS AS RELATED TO ELECTROLYTE CONCENTRATION AND TOXICITY

ISHS XXVI International Horticultural Congress: Environmental Stress and Horticulture Crops MECHANISM OF SALT TOLERANCE OF TWO OLIVE OLEA EUROPAEA L. CULTIVARS AS RELATED TO ELECTROLYTE CONCENTRATION AND TOXICITY

Aspectos biológicos dos estágios imaturos de Pseudodorus clavatus (Fabricius) (Diptera: Syrphidae) alimentados com Schizaphis graminum (Rondani) (Hemiptera: Aphididae) em diferentes temperaturas

Os aspectos biológicos de Pseudodorus clavatus (Fabricius), alimentada com Schizaphis graminum (Rondani), foram avaliados em diferentes temperaturas, visando obter informações para o controle biológico de afídeos. Ovos do predador foram individualizados em discos de folhas de sorgo, mantidos em placas de Petri, às temperaturas de 16, 19, 22, 25, 28 e 31 ºC e 12h de fotofase. Ninfas e adultos de S. graminum foram oferecidos ad libitum. Avaliou-se o número de ínstares, a duração e sobrevivência de cada ínstar, bem como das fases larval e pupal e do período de larva a adulto de P. clavatus. A temperatura base e a constante térmica também foram estimadas. A duração dos três ínstares de P. clavatus variou na razão inversa da temperatura. Houve redução das fases larval (14,9 para 4,9 dias) e pupal (17,2 para 4,2 dias) e do período de larva a adulto (31,7 para 8,8 dias) quando as larvas foram mantidas a 16ºC e 31ºC, respectivamente. As temperaturas bases estimadas foram de 10,8; 11,0 e 10,8ºC para as fases larval, pupal e para o período de larva a adulto, respectivamente. Em todas as temperaturas estudadas a sobrevivência do primeiro ínstar foi superior a 78% e do segundo e terceiro instares foi superior a 94%. As sobrevivências da pupa e do período de larva a adulto foram maiores a 22ºC e menores a 16ºC e 31ºC. O desenvolvimento completo de P. clavatus, alimentada com S. graminum, foi possível em todas as temperaturas testadas, que variaram de 16ºC a 31ºC.

Partial Purification and N-Terminal Amino Acid Sequencing of a β-1,3-Glucanase from Sorghum Leaves

A protein with an apparent molecular mass of 30 kDa that cross-reacts with barley glucanase antiserum was detected in healthy leaves of sorghum (Sorghum bicolor (L.) Moench). When sorghum leaves were infected with Exserohilum turcicum, the causal agent of leaf blight, the 30-kDa glucanase was substantially induced. The 30-kDa glucanase was partially purified from sorghum leaves using ammonium sulfate fractionation and anion exchange chromatography on DEAE-sephacel. The N-terminal amino acid sequence of the 30-kDa glucanase shows homology to glucanases of maize, barley, bean, soybean, tobacco and pea. The purified 30-kDa glucanase showed antifungal activity against Trichoderma viride.

A conserved 19-amino acid synthetic peptide from the carboxy terminus of phosphoenolpyruvate carboxylase inhibits the in vitro phosphorylation of the enzyme by the calcium-independent phosphoenolpyruvate carboxylase kinase.

Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 microm). This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize [Zea mays]) enzymes.

Molecular Cloning and Characterization of Starch Branching Enzyme in Sorghum Grain

The contribution of starch-branching enzyme(EC 2.4.1.18)to starch synthesis in developing sorghum(Sorghum bicolor)grains is very important. In the present work, the cDNA encoding sorghum starch branching enzyme was cloned, sequenced and characterized. This clone was termed SorBE and its accession number in public database is AF169833. SorBE consisted of an open reading frame of 2499 bp encoding a protein of 832 amino acids, with the putative molecular weight of 94.1 kDa. The upstream region of the SorBE mRNA was highly GC rich which caused the difficulty for the 5'-RACE cloning. This SorBE hybridized to mRNA of approximately 2,700 nucleotides whose accumulation was detected in developing sorghum seeds. Southern blot analysis of the sorghum leaf genomic DNA revealed SorBE was encoded by a single gene. Phylogenetic study indicated that this is a seed-specific type I starch branching enzyme.

Above‐ and below‐ground responses of C3–C4 species mixtures to elevated CO2 and soil water availability

AbstractWe evaluated the influences of CO2[Control, ∼ 370 µmol mol−1; 200 µmol mol−1 above ambient applied by free‐air CO2 enrichment (FACE)] and soil water (Wet, Dry) on above‐ and below‐ground responses of C3 (cotton, Gossypium hirsutum) and C4 (sorghum, Sorghum bicolor) plants in monocultures and two density mixtures. In monocultures, CO2 enrichment increased height, leaf area, above‐ground biomass and reproductive output of cotton, but not sorghum, and was independent of soil water treatment. In mixtures, cotton, but not sorghum, above‐ground biomass and height were generally reduced compared to monocultures, across both CO2 and soil water treatments. Density did not affect individual plant responses of either cotton or sorghum across the other treatments. Total (cotton + sorghum) leaf area and above‐ground biomass in low‐density mixtures were similar between CO2 treatments, but increased by 17–21% with FACE in high‐density mixtures, due to a 121% enhancement of cotton leaf area and a 276% increase in biomass under the FACE treatment. Total root biomass in the upper 1.2 m of the soil was not influenced by CO2 or by soil water in monoculture or mixtures; however, under dry conditions we observed significantly more roots at lower soil depths (> 45 cm). Sorghum roots comprised 81–85% of the total roots in the low‐density mixture and 58–73% in the high‐density mixture. CO2‐enrichment partly offset negative effects of interspecific competition on cotton in both low‐ and high‐density mixtures by increasing above‐ground biomass, with a greater relative increase in the high‐density mixture. As a consequence, CO2‐enrichment increased total above‐ground yield of the mixture at high density. Individual plant responses to CO2 enrichment in global change models that evaluate mixed plant communities should be adjusted to incorporate feedbacks for interspecific competition. Future field studies in natural ecosystems should address the role that a CO2‐mediated increase in C3 growth may have on subsequent vegetation change.

Relative time of redroot pigweed emergence affects dry matter partitioning

Abstract The partitioning coefficient is defined as the proportion of new dry matter partitioned among different plant parts. Partitioning coefficients can be used to model plant dry matter accumulation. In 1994 and 1995, field studies were conducted at two locations near Manhattan, KS, to determine the influence of density and relative time of emergence of redroot pigweed on dry matter partitioning to stem, leaves, and reproductive parts throughout the season. Redroot pigweed was grown with sorghum and in monoculture at densities of 2, 4, and 12 plants m−1 of row each year at each location. Dry matter partitioning during vegetative growth was not influenced by plant density. However, partition coefficients during the reproductive growth stage changed as a linear function of the time of pigweed emergence relative to the sorghum leaf stage. The later the emergence time relative to sorghum leaf stage, the higher the partitioning coefficient values for leaf (PCleaf) and stem (PCstem) and the lower the partit...

Simultaneous Temporal Progress of Sorghum Anthracnose and Leaf Blight in Crop Mixtures with Disparate Patterns

Field studies were conducted at Alupe in western Kenya in 1995 and 1996 to evaluate the efficacy of crop and species mixtures for the management of sorghum anthracnose (caused by Colletotrichum sublineolum) and leaf blight (caused by Exserohilum turcicum). The progress of these diseases developing simultaneously on a susceptible sorghum cultivar planted in inter- or intra-row mixtures of varying proportions with either maize or resistant sorghum was monitored. The effects of host type and mixture patterns on disease progress were compared by parameter estimates derived from fitted Lotka-Volterra competition equations and nonlinear logistic models. Competition coefficients were not significant and their confidence intervals included zero in most cases, suggesting that interactions between C. sublineolum and E. turcicum did not occur. Mixtures of the susceptible sorghum with either the nonhost maize or the resistant sorghum delayed the time when disease is first observed and reduced the rate of disease progress and carrying capacity for both anthracnose and leaf blight, with a more pronounced effect on the latter disease. The lower efficacy of mixtures in reducing anthracnose was attributed to an aggregated spatial pattern, coupled with higher rates of progress for this disease. Intra-row mixtures were more efficient than inter-row mixtures in reducing disease development in all years. The implications of these observations for the management of sorghum diseases under small-scale farming systems are discussed.

Development of seed cuticular waxes on bloomless mutants of Sorghum bicolor L. Moench

Development of seed cuticular waxes on wild-type Sorghum bicolor and isogenic bloomless (bm) mutants was analyzed using gas chromatography and mass spectrometry. The amount of cuticular waxes per seed dry weight of wildtype increased significantly between the soft dough and hard dough stage, and then decreased after the hard dough stage. Analysis of seeds from wildtype and two sorghum leaf wax mutants allowed identification of the first gene mutations that alter cuticular waxes of seeds. Total cuticular wax quantities on bm2 and bm4 seed surfaces varied between 37% and 75% of wild-type levels during development. Interestingly, bm4 had much higher proportions of short chain length wax constituents than wildtype and bm2 throughout development. These new mutant resources and knowledge about wax development presented here may prove useful for future studies to elucidate the role of cuticular waxes in pest resistance of grain.

A DNA-binding activity for the promoter of the gene encoding C 4 phosphoenolpyruvate carboxylase is modulated by phosphorylation during greening of the Sorghum leaf

Electrophoresis mobility shift assay (EMSA) identified nuclear proteins with binding activity to a 430 bp promoter fragment of the Sorghum C 4 phosphoenolpyruvate carboxylase gene ( SvC4). The DNA binding activities (two main retarded bands; PC1 and PC2) were high in nuclear extracts from etiolated leaves, decreased during greening and became very low or null in nuclear extracts from green leaves. This process was found to be mediated by phytochrome and was apparently irreversible since the DNA-binding activities were not restored in green plants kept in continuous darkness. The AT-rich region of the promoter fragment was identified to be the interaction domain of PC2. The detection of PC2 with EMSA was markedly reduced by preincubation of nuclear protein extracts with Mg-ATP or Mg-GTP and restored in the presence of a general protein serine/threonine-kinase inhibitor, K252a. The results suggested that the PC2 binding activity was modulated by phosphorylation during the greening process of the Sorghum leaf.

Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase

We have reported a simple method of determination of plasma oxalate using a Cl − and NO 3 − insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120–122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H 2O 2 generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 μmol/l. Analytic recovery of added oxalate in plasma was 89.5±4.1% (mean±S.D.). The within and between day CV for plasma oxalate measurement were <9.37 and <11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 μmol/l. The method is not only free from interference by plasma Cl − and NO 3 − but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.