Patricia L. Herman

A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules

The GL1 gene is required for the initiation of differentiation of hair cells (trichomes) on the crucifer, Arabidopsis thaliana. This gene has been localized to a 4.5 kb DNA fragment by molecular complementation of gl1 mutants. DNA sequence analysis has shown that the protein encoded by GL1 contains a Myb DNA-binding motif. Southern analysis and subsequence analysis of isolated lambda clones has established that GL1 is a member of an extensive myb gene family in Arabidopsis. The putative GL1 promoter directs the expression of the GUS reporter gene in non-trichome-bearing structures that appear to be stipules. This pattern of expression suggests that GL1 may control the synthesis of a diffusible signal that activates the developmental pathway for trichome differentiation.

Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies.

The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast-encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined.

Trichome Development in Arabidopsis thaliana. II. Isolation and Complementation of the GLABROUS1 Gene.

We are using the formation of trichomes in Arabidopsis thaliana as a model system to study gene expression during cellular differentiation. To initiate the molecular characterization of this system, we tagged and isolated a gene that is specifically required for the development of the specialized trichome cell. We confirmed the identity of this gene, GLABROUS1 (GL1), by complementation. These results demonstrate that a crucial gene in a plant developmental pathway can be successfully identified by complementation.

Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA

Hundreds of mutants with defects in a variety of physiologically important functions, such as photosynthesis, respiration, flagellar motility, phototaxis, circadian rhythms and the cell cycle, have been isolated from cultures of Chlamydomonas reinhardtii. In only a few cases have the genes responsible for these mutations been cloned and sequenced. The development of efficient methods for transformation with nuclear genes [7] has allowed the recent demonstration of gene isolation through genomic complementation with a pooled library of C. reinhardtii DNA [9]. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed (ordered) cosmid library of 11,280 individual clones contained in the separate wells of 120 microtiter plates. The average insert size is ca. 38 kb. PCR analysis of five sequenced nuclear genes present in the Chlamydomonas library revealed a range from two copies for the α2 and β2 tubulin genes to at least seven copies for the agininosuccinate lyase gene. Overall, these five clones were represented an average of >-3.4 times in the library. Thus, the probability that any one particular nuclear gene of < 1000 bp will be found in the library is >-97%, and the probability that a gene of ca. 10 000 bp will be found in the library is ca. 92%. Rapid screening methods with cosmid DNAs pooled from individual microtiter dishes have been applied successfully. Bacteria containing clones of the argininosuccinate lyase gene have been identified through genomic complementation of a Chlamydomonas mutant bearing an inactive arginnosuccinate lyase gene.We are using the nomenclature of ‘indexed’ library versus ‘ordered’ library to avoid confusion of this library with a library of ordered contigs.

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.

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.

Sequence of a psaC gene from the cyanobacterium Synechococcus sp. PCC 6301.

The psaC gene encodes PsaC, the apoprotein for the terminal iron-sulfur clusters, FA and FB, in the PSI reaction center of cyanobacteria, algae, and higher plants. PsaC functions as a membrane-bound oxidoreductase, accepting electrons from the Fx iron-sulfur cluster located on the PsaA/ PsaB heterodimer and donating them to the soluble electron transfer proteins Fd and flavodoxin (reviewed in Bryant, 1992). The objective of our work is to clarify the role of PsaC in linear and cyclic electron transfer. The experimental organism is Synechococcus sp. PCC 6301, a unicellular, freshwater cyanobacterium that is used extensively for physiological, biochemical, and spectroscopic studies of photosynthesis. The PSI complex of Synechococcus sp. PCC 6301 is widely used in structural and functional studies of the FA and FB ironsulfur clusters (Parrett et al., 1990). We became interested in the psaC sequence of Synechococcus sp. PCC 6301 because there are subtle differences in the electron paramagnetic resonance spectra of the FA and FB iron-sulfur clusters between cyanobacteria and higher plants. This suggests that there may be a correlation with structure: in all organisms studied thus far, the higher-plant PsaC protein differs from the cyanobacterial PsaC protein in two regions: at position 37, higher plants contain a Lys whereas cyanobacteria contain a neutral amino acid (Gly or Ala), and at positions 70 and 71, dicots contain a Trp-His pair, monocots contain a Gly-Pro pair, and cyanobacteria contain a GlyAla pair. No sequence for the PsaC protein of Synechococcus sp. PCC 6301 is available, even though entire sequences are available for PsaD and PsaE and partial sequences are available for PsaL and PsaF. This information is relevant because

Acetylcholinesterase Activity in Plants

Acetylcholinesterase, AChE, (E.C.3.1.1.7), has been reported to be present along with acetylcholine, ACh, in many plant species (Hartmann and Gupta, 1989; Tretyn and Kendrick, 1991). A defined role for ACh in plant cellular processes is still unclear but suggestions have been made that it may act as a second messenger in various processes (Hartmann and Gupta, 1989). We have presented in this study data supporting: a) the presence and enrichment of AChE in guard cells of stornata in three different plant species, and b) the possible involvement of ACh in the regulation of stomatal movements.