John C. Osterman

Calibration of the Minolta SPAD-502 leaf chlorophyll meter

Use of leaf meters to provide an instantaneous assessment of leaf chlorophyll has become common, but calibration of meter output into direct units of leaf chlorophyll concentration has been difficult and an understanding of the relationship between these two parameters has remained elusive. We examined the correlation of soybean (Glycine max) and maize (Zea mays L.) leaf chlorophyll concentration, as measured by organic extraction and spectrophotometric analysis, with output (M) of the Minolta SPAD-502 leaf chlorophyll meter. The relationship is non-linear and can be described by the equation chlorophyll (μmol m−2)=10(M0.265), r2=0.94. Use of such an exponential equation is theoretically justified and forces a more appropriate fit to a limited data set than polynomial equations. The exact relationship will vary from meter to meter, but will be similar and can be readily determined by empirical methods. The ability to rapidly determine leaf chlorophyll concentrations by use of the calibration method reported herein should be useful in studies on photosynthesis and crop physiology.

Aspergillus niger mutants with increased glucose oxidase production

SummaryAspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.

Formate dehydrogenase in Arabidopsis thaliana: characterization and possible targeting to the chloroplast

Formate dehydrogenase (E.C. 1.2.1.2) is a mitochondrial-localized NAD-requiring enzyme in green plants. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region, which is predicted to target chloroplasts as well as mitochondria. The specific of activity of the enzyme in isolated chloroplasts suggests it is targeted to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and K(m) values for formate and NAD(+) were determined to be 10 mM and 65 µM, respectively; the K(i) for NADH was 17 µM. We conclude that formate dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play a role in whether cellular formate is assimilated or dissimilated.

Formate dehydrogenase in Arabidopsis thaliana: overexpression and subcellular localization in leaves

Abstract Formate dehydrogenase (FDH; EC 1.2.1.2) is a NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide in the mitochondria of higher plants. Sequence analyses and other preliminary experiments suggested that FDH might also be targeted to the chloroplasts of Arabidopsis thaliana and other plant species. In the present study, transgenic Arabidopsis and tobacco plants that overexpress Arabidopsis FDH were produced. The FDH specific activity in the leaf tissue of the transgenic plants increased an average of 4.5-fold for Arabidopsis and 31.5-fold for tobacco. Immunodetection and enzyme assays of intact chloroplasts fractionated from the leaves of transgenic tobacco plants suggested that Arabidopsis FDH is present in the chloroplast. Immunogold labeling of Arabidopsis and tobacco detected FDH in both the mitochondria and chloroplasts of the leaf cells. Enzyme assays that were performed to confirm the specificity of Arabidopsis FDH for the NAD cofactor suggested that NADPH was an inhibitor of NAD + dependent formate oxidation.

Molecular analysis of the ADH1-Cm allele of maize

The Adh1-Cmallele and each gene in the Adh1-FCmduplication have been cloned and restriction-mapped. Of the Cmallele 6 kb was sequenced. A single amino acid substitution of aspartate for tyrosine at residue 52 accounts for the altered enzymatic properties of the Cmprotein. Comparison of the nucleotide sequence to that of Adh1-1F and Adh1-1S shows structural and restriction site polymorphisms in the 3′ flanking DNA. Cmlacks the insertion sequence present in 1F and 1S and contains a complex sequence composed of two direct repeats and an inverted repeat. The two genes of the duplication allele have similar restriction maps to Cmand each other.

Assessing modulation of stromal and thylakoid light-harvesting complex-II phosphatase activities with phosphopeptide substrates

The study of the light-harvesting complex II (LHC-II) phosphatase activity has been difficult due to the membrane association of its substrate. Thylakoid membranes labeled with [γ-32P]ATP were incubated with chymotrypsin, releasing phosphopeptides which served as labeled substrates for LHC-II phosphatase. Utilizing these phosphopeptides as substrates, protein phosphatase activities have been identified in both the thylakoid membrane and the stromal fraction. The thylakoid-bound phosphatase was liberated from the membrane with a sub-solubilizing concentration of Brij 35. The membrane and the stromal protein phosphatases were inhibited by NaF and EDTA, but not inhibited by microcystin-LR. The stromal phosphatase differed from the membrane phosphatase in pH optimum, in its lack of inhibition by molybdate ions, and by its response to magnesium and manganese ions. Using the soluble chymotryptic peptide substrate, the effect of light on pea thylakoid-bound LHC-II phosphatase activity was also assessed. Incubation of the thylakoid membranes in the light caused a 35% inhibition of LHC-II phosphatase activity. The inhibition was diminished by the addition of DCMU. Addition of 10 mM dithiothreitol stimulated the activity in darkness and obviated the inhibition when exposed to light. These studies suggest that positive or negative regulation of the LHC-II phosphatase activity is possible in vivo.

Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.

Kinetic behavior of the Arabidopsis thaliana leaf formate dehydrogenase is thermally sensitive

Two previous kinetic studies on the Arabidopsis thaliana leaf NAD-dependent formate dehydrogenase (EC 1.2.1.2) have demonstrated two very different sets of Km values for the formate and NAD+ substrates. We examined the kinetics of the enzyme partially purified from a leaf extract by gel-filtration desalting and chromatography on DEAE-cellulose, as well as by isolation of a mitochondria-enriched fraction obtained by differential centrifugation. Both of these methods produce a formate dehydrogenase enzyme with the higher Km values of approximately 10 mmol/L formate and 75 mumol/L NAD+. The kinetic properties of the Arabidopsis formate dehydrogenase expressed to high levels in transgenic tobacco plants were also those of the high Km form. The high Km form of the enzyme converted to a low Km form by heating for 5 minutes at 60 degrees C. An Arrhenius plot of the activity during the heating process was linear, indicating that the heating did not cause alterations in either the active site or the thermal dependence of the catalytic reaction. We conclude that the native form of the formate dehydrogenase probably resembles the form with the higher Km values. Heating seemingly converts this native enzyme to the molten globule state and cooling results in formation of a non-native structure with altered kinetic properties.

Temperature sensitivity as a general phenomenon in a collection of chlorophyll-deficient mutants of sweetclover (Melilotus alba)

A collection of chlorophyll (Chl)-deficient mutants of sweetclover (Melilotus alba) with defects in eight nuclear loci were grown at 17 or 26° C. Plants grown at either temperature were examined for Chl content, Chla/b ratio, expression of the light-harvesting complex II (LHC-II) apoproteins, and protochlorophyllide (Pchlide) biosynthetic capacity. Except for thech4 mutant, the parental strain and all mutants accumulate more Chl when grown at 26° C than at 17° C. Thech5 mutants, lacking Chl b under any growth condition, and thech12 mutant showed little temperature-dependent phenotypic plasticity, whereas this was a marked phenomenon in the other mutants. Thech10 andch11 mutants demonstrated extreme temperature sensitivity with regard to the production of Chlb and the Chlb-binding LHC-II apoproteins. When excised trifoliolates were supplemented with exogenously supplied δ-aminolevulinic acid, only thech4 mutant was markedly impaired in the ability to produce Pchlide. These data indicate that temperature-sensitive phenotypic plasticity is a common phenomenon of chlorophyll-deficient mutants and substantiate that only a minority of Chl-deficient mutants is impaired in the biosynthesis of Chl.

An allele of the Prot locus in maize is a variant for the site of protein processing

An allele of theProt locus, which encodes a major globulin of the maize scutellum, is a variant for a site of protein processing. Segregation analysis and recombination mapping indicate that the variant is an allele of theProt locus. DesignatedProt-V, this allele specifies three polypeptides, V1, V2, and V3. The V1 polypeptide is incompletely processed during the proteolytic processing step catalyzed by the product of theMep locus. Cyanogen bromide cleavage studies support the precursor-product relationship between V1 and V2. The V1 product is shortened with respect to other PROT′ proteins and it is postulated that the normal site of MEP processing has been removed by this foreshortening.