Hidemasa Imaseki

Ethylene Stimulates Endoreduplication But Inhibits Cytokinesis in Cucumber Hypocotyl Epidermis

The effects of ethylene on cell division are generally considered inhibitory. In this study, we demonstrate that transient ethylene exposure, while suppressing cytokinesis, stimulates DNA synthesis. We monitored DNA synthesis and cytokinesis in the epidermis of cucumber (Cucumis sativus) hypocotyls, an organ whose post-germination development involves strictly limited cell division. During exposure to ethylene, DNA synthesis, assessed by the incorporation of the thymidine homolog 5-bromo-2′-deoxyuridine, was detected in 20% of the epidermal cells, whereas DNA synthesis was nearly undetectable in normal air. Cytofluorometric analysis of nuclei in affected cells showed an up to 8-fold increase in DNA content. During this time, new cell plate formation was not detected. However, shortly after ethylene was removed, DNA content was rapidly restored to 2C (diploid) levels in all cells, and new cell plate formation dramatically increased. These results demonstrate that ethylene promotes DNA synthesis and its endoreduplication but inhibits cytokinesis, thereby maintaining some cells in G2 phase.

Identification of a Basic Glycoprotein Induced by Ethylene in Primary Leaves of Azuki Bean as a Cationic Peroxidase

Ethylene causes the accumulation of seven different proteins (each designated AZxx according to its molecular mass, xx in kD) in excised primary leaves of azuki bean (Vigna angularis) (F. Ishige, H. Mori, K. Yamazaki, H. Imaseki [1991] Plant Cell Physiol 32:681–690). A complementary DNA encoding an ethylene-induced basic glycoprotein, AZ42, from azuki bean was cloned and its complete nucleotide sequence was determined. Characterization of the cDNA was accomplished by monitoring expression of an immunoreactive protein in Escherichia coli that harbored the cDNA and by the identification of a partial amino acid sequence that was the same as that determined from the purified protein. An open reading frame (1071 base pairs) in the cDNA encoded a protein of 357 amino acids with a molecular mass of 39.3 kD. The amino acid sequence contained three regions that are highly conserved among peroxidases from eight different plants. Purified AZ42 exhibited peroxidase activity. The basic glycoprotein induced by ethylene was identified as a cationic isozyme of peroxidase. The corresponding mRNA was not present in leaves that had not been treated with ethylene, but it appeared after 1 h of treatment with ethylene and its level increased for the next 15 h. Accumulation of the mRNA was also induced after wounding or treatment with salicylate. The wound-induced increase in the level of the mRNA was suppressed by 2,5-norbornadiene, but the salicylate-induced increase was not.

DNA sequence requirement of a TATA element-binding protein from Arabidopsis for transcription in vitro

We have analyzed the DNA sequence requirements for the functioning of TATA elements by examining the transcriptional activities associated with 24 promoters, including representatives of each of the 21 point mutations in the consensus sequence from plants, TATATATA, in a HeLa in vitro system and in a chimeric in vitro system in which human TATA-binding protein (hTBP) was replaced by purified TBP of Arabidopsis (aTBP-1). Although the relative transcriptional activities varied among these promoters, both systems gave virtually identical results. Among the mutant TATA elements, those with the sequences TAGAGATA and GAGAGAGA had undetectable activity. The rest had activities that ranged from 7% to 130% of the activity associated with the consensus element. These results suggest the functional conservation of TBP between plants and animals.

Chapter 9 – Control of ethylene synthesis and metabolism

Ethylene is amongst the best-known plant hormones because of the extensive biochemical and molecular studies on its biosynthesis and its regulation. It is synthesized in both higher plants and microorganisms, but via different biosynthetic pathways. One of the characteristic features of ethylene production in higher plants is that the rate of production frequently changes both during normal development and in response to environmental stimuli. From seed germination to the early stages of seedling growth, a moderate level of ethylene production is generally observed; particularly in the apical portion and at the nodes. However, during later vegetative growth, the rate of production decreases to a very low level. Moreover, ethylene production from tissues is influenced by environmental stimuli. When tissues are physically wounded or stressed, infected by pathogens, or injured by toxic chemicals, a large increase in ethylene production occurs in living tissue near the damaged cells. Extensive studies on ethylene production in higher plants have firmly established the hormonal function of the gas during plant development and in responses to environmental stimuli. However, the biological significance of microbial ethylene production is not known.

Monomeric and dimeric forms and the mechanism-based inactivation of 1-aminocyclopropane-1-carboxylate synthase

Among ACC synthase preparations of various origins, i.e. those from tomato and winter squash fruits as well as those expressed by E.coli from cDNAs for tomato and winter squash ACC synthase, only the enzyme from tomato fruit tissue existed in a monomeric form, whereas the others in a dimeric form. The monomeric tomato ACC synthase was much less sensitive to the mechanism-based inactivation than the dimeric forms of ACC synthase. We suggest that ACC synthases have a property to form a dimer, but in tomato fruit tissue some modification takes place to the enzyme protein, which makes it remained as a monomer and less sensitive to the mechanism-based inactivation.

Ethylene biosynthesis: Methionine as an in-vivo precursor of ethylene in auxin-treated mungbean hypocotyl segments

SummaryEthylene production was induced in excised hypocotyl segments of etiolated mungbean seedlings in response to exogenous auxin. 1,2,3,4-14C-Methionine was efficiently incorporated into C2H4, although cold methionine added at substrate level did not enhance C2H4 production. Incorporation of labeled glucose into C2H4 was reduced when hypocotyl segments were incubated with cold methionine and homoserine, but the rate of labeling of CO2 was not affected. Feeding of labeled glucose to segments resulted in production of labeled methionine both in the presence and the absence of auxin, and auxin did not affect rate of methionine synthesis from glucose. The decrease in the amount of endogenous methionine during auxin treatment approximated the amount of C2H4 produced. The timecourse pattern of incorporation of radioactivity from labeled methionine into C2H4 during removal or re-addition of auxin was very similar to that of C2H4 production. The role of endogenous methionine as a C2H4 precursor is discussed.

A furcatin hydrolyzing glycosidase of Viburnum furcatum Blume.

Abstract Some properties of a glycosidase obtained from the leaves of Viburnum furcatum are described. The Viburnum glycosidase hydrolyzes furcatin into the aglycone, p-vinylphenol and apiosyl-1,6-glucose and it seems that the glycosidase requires apiosyl-1,6-glucoside bond for its action. Since the hydrolytic activity of the glycosidase is inhibited by a low concentration of heavy-metal ion or by some typical SH inhibitors, the SH group of the glycosidase may play an important part, in the reaction. Experiments with a mixture containing alcohol show that the Viburnum glycosidase transfers apiosylglucosyl residue of furcatin to alcohol forming an alkyl apiosyl-glucoside, and the effects of alcohol on both the hydrolysis of furcatin and the transglycosidation reaction were examined.

Accurate transcription of plant genesin vitro using a wheat germ-chromatin extract

We describe here anin-vitro system for accurate transcription initiation of plant promoters. The system consists of a wheat-germ chromatin extract, which serves as a source of RNA polymerase II and basic transcription factors, substrates, and exogenously added DNA templates. Both linear and circular DNA are equally effective as templates, but optimal conditions for their transcription differ slightly. Specific initiation at the promoter of a linear template is determined by size analysis of the transcript on a polyacrylamide gel containing 8 M urea. In the case of circular DNA template, the initiation frequency is measured by the amount of specific RNA transcribed from a G-free sequence (Sawadogo and Roeder, 1985) in a reaction mixture without GTP. We believe that ourin-vitro system detailed here will be useful for the elucidation of transcription mechanisms in plants.

A proteinaceous inhibitor of ethylene biosynthesis by etiolated mungbean hypocotyl sections.

SummaryA protein which reversibly inhibits auxin-induced ethylene synthesis has been isolated and purified from hypocotyls of etiolated mungbean (Phaseolus aureus Roxb.) seedlings. The molecular weight of the inhibitor was estimated to be 112 000 by gel filtration and polyacrylamide gel-electrophoresis. When treated with sodium dodecylsulfate, the inhibitor gave on polyacrylamide gel-electrophoresis a single band corresponding to a molecular weight of 56 000, indicating that it consisted of two subunits with identical molecular weight. The inhibitor does not degrade nor bind indole-3-acetic acid, and has no peroxidase activity.

Biosynthesis of ethylene and its regulation in plants

Ethylene production rate changes rapidly during the life cycle of higher plants. Such change results from rapid induction of the rate-limiting enzyme in the biosynthetic pathway and the rapid inactivation of the enzyme. ACC synthase has been shown to be the rate-limiting enzyme. Auxins and tissue wounding stimulate ethylene production by specifically inducing the formation of ACC synthase which is then quickly inactivated within cells (apparent half life of 25 min). Thus, when synthesis of ACC synthase is slowed or stopped, rate of ethylene production quickly slows or stops. Synthesis of the enzyme is also controlled by other plant hormones including cytokinins, abscisic acid and ethylene. To understand the mechanisms regulating the synthesis of this enzyme at the molecular level, we have attempted to purify the enzyme from auxin-treated mung bean stems. To date this has been unsuccessful because of the unusual instability of the enzyme. Instead, we have purified the wound-induced enzyme from winter squash mesocarp as well as raised its specific antibody. Immunochemical analysis of western blots of crude proteins and fluorography of in vitro translation products of poly (A)RNA indicated that enzyme protein and mRNA accumulate due to wound stimulus. The regulatory mechanism of ACC synthase induction is discussed.