Donald E. Fosket

Oryzalin, a dinitroaniline herbicide, binds to plant tubulin and inhibits microtubule polymerization in vitro

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10-8 M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10-7 M oryzalin stopped migration abruptly. Oryzalin (1.0·10-7 M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Pauls scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (Ki) of 2.59·106 M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [14C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (Kapp) of 1.19·105 M-1. Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.

Inhibition of Plant Microtubule Polymerization in vitro by the Phosphoric Amide Herbicide Amiprophos-Methyl

The phosphoric amide herbicide amiprophos-methyl (APM) produced a concentration-dependent inhibition of taxol-induced rose microtubule polymerization in vitro. Parallel studies on taxol-induced assembly of bovine brain microtubules showed no effect of APM at a concentration ten times that required to give complete inhibition of rose microtubule assembly. The data indicate that (i) APM is a specific and potent antimicrotubule drug and (ii) APM directly poisons microtubule dynamics in plant cells, rather than indirectly depolymerizing microtubules through a previously proposed mechanism involving deregulation of intracellular calcium levels.

The biochemistry of compounds with anti-microtubule activity in plant cells

The experimental use of anti-microtubule compounds has revealed essential functions of microtubules in plant cytoskeletal arrays, including the pre-prophase band, the mitotic and meiotic spindles, the phragmoplast, and the cortical array. The most commonly used plant microtubule depolymerization compounds are colchicine, and several synthetic herbicides belonging to three different chemical classes, the dinitroanilines, phosphoric amides, and N-phenyl carbamates. Taxol, a secondary plant product, is the only drug found to promote the polymerization of plant microtubules. This paper summarizes our current understanding of the biochemical interactions of colchicine, anti-microtubule herbicides, and taxol with plant tubulin and microtubules.

The isolation, characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.)

Two divergent β-tubulin genes (designated Sβ-1 and Sβ-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii β-tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different β-tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of β-tubulin genes (thus far undetected) exist in the soybean genome. The Sβ-1 and Sβ-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode β-tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with β-tubulins from several organisms showed that they are most homologous to Chlamydomonas β-tubulin (85–87%), with lesser degrees of homology to β-tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of Sβ-1 and Sβ-2 are as divergent from each other as they are from the Chlamydomonas β-tubulin. The amino acids at the diverged positions in Sβ-2 are nearly all conservative substitutions while in Sβ-1, 18 of the 69 substitutions were non-conservative. Both soybean β-tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas β-tubulin genes. Codon usage in the two soybean β-tubulins is remarkably similar (D2=0.87), but differs from codon usage in other soybean genes.

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Pauls scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (Ki) of 1.4·10-4 M for rose tubulin and an apparent Ki=8.8·10-7 M for brain tubulin. The binding of [3H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [3H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·102 M-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·106 M-1 and r=0.45 at 37°C. The binding of [3H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.

Developmental modulation of tubulin protein and mRNA levels during somatic embryogenesis in cultured carrot cells

The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somatic embryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and torpedo/plantlet stages. The increase during the torpedo/plantlet stage was correlated with the increase in cell size that occurred during the latter stages of embryogenesis. The cellular levels of tubulin mRNA were determined by Northern blot analysis, using labeled probes derived from soybean α- and β-tubulin genomic sequences, cloned in the vectors pSP64 and pSP65. This analysis demonstrated that the levels of tubulin-gene transcripts varied with the tubulin-protein levels. Cell-free translation of polyadenylated RNA, followed by immunoprecipitation with an anti-tubulin antiserum, established that these transcripts represented functional tubulin mRNA. These results indicate that MT formation in early embryogenesis is controlled by factors other than the availability of tubulin, but that MT formation later in embryogenesis is coordinated with concomitant changes in tubulin-gene transcription and in the size of the total tubulin-heterodimer pool.

Limited expression of a diverged β-tubulin gene during soybean (Glycine max [L.] Merr.) development

We examined the developmental expression of a diverged soybean β-tubulin gene (designated sb-1), which had been cloned and sequenced previously. A probe specific for the sb-1 gene was constructed from the 3′ transcribed untranslated sequence. As a control, a more general probe for β-tubulin genes and their transcripts was constructed from a highly conserved region of the third exon of another soybean β-tubulin gene, sb-2. Poly(A)+ RNA, extracted from various soybean tissues and organs, was probed alternatively with the sb-1 gene-specific probe and with the generic β-tubulin probe. Levels of β-tubulin transcripts recognized by the generic probe differed by a factor of approximately 3 in the different tissues and organs and varied with the state of organ development. Highest levels were found in young, unexpanded leaves and they decreased as leaf maturation occurred. In contrast, transcripts of sb-1 were nearly undetectable in young leaves, and they increased as leaf maturation occurred. Levels of sb-1 transcript were low in all organs of the light-grown plant examined, except the hypocotyl, where they were approximately 10-fold higher. However, the highest levels of sb-1 transcripts were observed in elongating hypocotyls of etiolated seedlings. Exposure of six-day-old etiolated seedlings to light for 12 hours halted further hypocotyl elongation and brought about a dramatic, nearly 100-fold, decrease in the steady-state level of sb-1 transcripts.

Induction of carotenogenesis in cultured cells of Lycopersicon esculentum

Abstract The bioregulator 2-(4-chlorophenyl-thio) triethylamine (CPTA) which is known to induce lycopene formation and chromoplast differentiation in immature tomato fruits, was tested for its ability to bring about carotenogenesis in suspension cultures of tomato cells ( Lycopersicon esculentum cv. EP-7). Untreated dark-grown cultured tomato cells contain low levels of carotenoids, of which lycopene and β-carotene are the most abundant. The addition of CPTA to the culture medium brought about over a 60-fold increase in their total carotenoids during a 14-day culture period. The carotenoid content of the cells increased at a constant rate throughout the log phase of growth and this increase was brought about primarily by the accumulation of lycopene. When CPTA was given to logarithmically growing cells, carotenoid accumulation began after a 3-h lag period. This CPTA-heximide increase in carotenoids could be blocked by simultaneous cycloheximide (CH) treatment at concentrations of this drug which were shown to block [ 3 H] leucine incorporation into protein.

Hormonal regulation of growth in cultured plant cells

SummaryThe group of naturally occurring plant hormones known as the cytokinins are defined by their ability to stimulate cell division in mature, differentiated, mitotically inactive cells in tissue culture. Evidence from the literature suggests that cytokinins play a specific role in regulating the progress of a plant cell through its division cycle;. the hormone appears to trigger the transition from G2 to mitosis. However the cytokinins are capable of evoking an array of physiological and developmental responses (many of which do not involve cell division) from different plant tissues and organs. One biochemical effect of the cytokinins is a dramatic and rapid stimulation of polyribosome formation in cultured soybean, cells which require these hormones for growth. Stationary-phase soybean cells, transferred to a medium containing a cytokinin, double in cell number within 36 hr, but when transferred to a medium of the same composition lacking a cytokinin, they do not grow., In vivo labeling with [35S] methionine and slab-gel electrophoresis demonstrated that cytokinin brings about qualitative changes in the spectrum of proteins synthesized by soybean cells which precede hormone-induced cell division. We have shown that cytokinin-induced polyribosome formation is the result of an effect of the hormone on protein synthesis at the translational level. We propose that, in the absence of the hormone, certain genes are transcribed but their messengers are not translated. These include mRNAs for specific cell division proteins. Cytokinins act as permissive factors, allowing cells to complete a genetically programmed sequence of events which was initiated by other factors.

Inhibition of plant cell proteolytic activities that degrade tubulin

The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Pauls scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.