Edward A. Funkhouser

Cloning of a cDNA for a chitinase homologue which lacks chitin-binding sites and is down-regulated by water stress and wounding.

A cDNA clone (pLP6) of a gene which is repressed under water deficit was isolated from a loblolly pine (Pinus taeda L.) cDNA library and characterized. The predicted polypeptide encoded by pLP6 bears strong resemblance to a number of Class I chitinases. Howerver, LP6 lacks most of the amino-terminal and, consequently the signal peptide, cysteine-rich chitin-binding domain and glycine/proline-rich ‘hinge’ region, diagnostic of Class I chitinases, are absent. Although the cDNA is similar in size to its mRNA, the long open reading frame encoding the LP6 protein commences halfway through the mRNA, implying a 5′-untranslated region of over 700 nucleotides. Subfragments from the 5′ end of pLP6 hybridize to the same mRNA as do probes consisting of the entire cDNA. Reverse transcription (RT)-PCR experiments confirm that the cDNA derives from a single mRNA molecule. Analysis of the 5′-UTR revealed six upstream open reading frames and four inverted repeat structures. Expression of the pLP6 gene is repressed by water deficit stress and wounding. Possible functions and origin of this gene are discussed.

Characterizing gene responses to drought stress in fourwing saltbush (Atriplex canescens (Pursh.) Nutt.)

New techniques in molecular biology can be used to characterize genes whose expression is induced by drought stress. These techniques can be used to understand responses of range plants to environmental stresses at the biochemical and molecular level. For example, they can be used to characterize genes that respond to drought stress conditions in the native shrub Atriplex canescens (Pursh.) Nutt. Complementary DNA (cDNA) libraries constructed from drought-induced messenger RNA (poly A+ RNA) were used to characterize genes which are associated with the stress response. A cDNA library from A. canescens was prepared. This library from stressed shrubs was differentially screened with radiolabeled cDNA probes from stressed and nonstressed shrubs, and apparent drought-induced clones were identified. This is the first report of molecular characterization of drought responsive genes in four-wing saltbush. The identification of genes specific to responses to drought stress could provide a basis for understanding drought tolerance in this important range species.

Induction and synthesis of nitrate reductase in Chlorella vulgaris

Assimilatory nitrate reductase is an inducible, eukaryotic enzyme that responds to a variety of environmental cues. When higher plants and green algae are grown with ammonia as a nitrogen source, low levels of nitrate reductase activity are present. Transfer to nitrate-containing medium is accompanied by substantial increase of nitrate reductase activity. Here it is shown immunologically that, in the green algae Chlorella vulgaris, nitrate reductase protein is over-produced as activity appears during induction. Immunoreactive protein is also found in cells grown on ammonia. Low levels of translatable mRNA for nitrate reductase are present in ammonia-grown cells. These data suggest that: (i) nitrate reductase appearance is controlled primarily on a transcriptional level, but that transcription is not completely halted under repressing conditions; (ii) there is an overproduction of nitrate reductase protein early during the induction period as previously suggested; and (iii) nascent protein, from in vitro translation, is of approximately the same molecular size as the nitrate reductase subunit and therefore little posttranslational modification is necessary to generate the functional enzyme. Insertion of cofactors and assembly are probably the only post-translational events.

Changes in nitrate reductase protein during loss and gain of nitrate reductase activity in Chlorella vulgaris

Abstract Nitrate reductase (NADH) (NADH: nitrate oxidoreductase, EC 1.6.6.1) in Chlorella vulgaris exists, in vivo, in at least two forms. One is active, and the other, a reversibly inactived form, accumulates when nitrate assimilation is inhibited. This inactive form converts, in vivo, to the active form under other culture conditions which promote nitrate metabolism. This inactive form can also be activated in vitro by oxidation with reagents such as ferricyanide. The only inactive form which is characterized is a cyanide complex of the reduced enzyme that also can be formed in vitro. The possibility of other inactive forms has been suggested, but none has been reported. In the present study, active and ferricyanide-activatable nitrate reductase activities were determined in crude extracts from cells grown under very different conditions. Nitrate reductase protein in these extracts was determined by rocket immunoelectrophoresis. Conditions which lower the level of active and activatable nitrate reductase activites concomitantly lower nitrate reductase protein. Similarly, when total activities increase, there is a corresponding increase in nitrate reductase protein. Since all changes in nitrate reductase activity may be accounted for by corresponding changes in nitrate reductase protein, the cyanide complex of nitrate reductase is the only major inactive form of nitrate reductase in Chlorella .

Continuous determination of effect of temperature on enzymic activities

An electronic temperature controller was used to continuously change the temperature of cuvettes during enzymic assays. A microprocessor-controlled spectrophotometer continuously collected absorbance, time, and temperature data. Activation energies were calculated from Arrhenius plots of these data.

Endopeptidase Activity in Cotton Cotyledons During Development

Summary Protease levels in cotton cotyledons were monitored from germination until abscission. When assayed by the radial diffusion assay, there were three peaks of activity; one at germination, one at the time of autotropy, and one at senescence.