Bernard Rubinstein

Regulation of cell death in flower petals

The often rapid and synchronous programmed death of petal cells provides a model system to study molecular aspects of organ senescence. The death of petal cells is preceded by a loss of membrane permeability, due in part to increases in reactive oxygen species that are in turn related to up-regulation of oxidative enzymes and to a decrease in activity of certain protective enzymes. The senescence process also consists of a loss of proteins caused by activation of various proteinases, a loss of nucleic acids as nucleases are activated, and enzyme-mediated alterations of carbohydrate polymers. Many of the genes for these senescence-associated enzymes have been cloned. In some flowers, the degradative changes of petal cells are initiated by ethylene; in others, abscisic acid may play a role. External factors such as pollination, drought and temperature stress also affect senescence, perhaps by interacting with hormones normally produced by the flowers. Signal transduction may involve G-proteins, calcium activity changes and the regulation of protein phosphorylation and dephosphorylation. The efficacy of the floral system as well as the research tools now available make it likely that important information will soon be added to our knowledge of the molecular mechanisms involved in petal cell death.

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.

Activities of nucleases in senescing daylily petals

Abstract The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs.

Indoleacetic-acid-enhanced chloride uptake into coleoptile cells

SummaryThe enhancement by indoleacetic acid (IAA) of 36Cl- uptake into Avena coleoptile sections was used to study the effects of a hormone on a membrane-controlled phenomenon. Compared to sections in phosphate buffer only, Cl- content of the cells increases 15 min after addition of IAA; the promotion is seen only with growth-active auxins and is saturated at 3 μM IAA. The percent enhancement by IAA is the same over a wide range of Cl- concentrations. The hormone effect is not observed at ice-bath temperature and is not correlated with growth or water movement into the cells. IAA does not influence the movement of Cl- in the section. While auxin must be present within the tissue in order to maintain the enhancement, there is no relationship between the total amount of auxin and the accelerated Cl- uptake that results. A polarity in the auxin effect is implied since only apical applications of IAA promote Cl- uptake.

Light-stimulated transplasmalemma electron transport in oat mesophyll cells

Abstract Transplasmalemma redox activity has been detected at the peeled surface of oat ( Avena sativa L. cv. ‘Garry’) leaf segments by measuring the reduction of the non-permeating oxidant, ferricyanide. This activity is stimulated 70–100% by white light, but light has no effect when exogenous NADH is used as an electron donor. The light stimulation of transplasmalemma ferricyanide reduction is prevented by 50 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU). Blue and red light are as effective as white light in promoting redox activity, while green is one-third as active. These data suggest a role for photosynthesis. Respiratory poisons decrease redox activity in the dark, but a light-induced stimulation is still observed. The stimulated rate decays in the dark with a half-life of 3 min in the presence or absence of respiratory poisons, implicating a chemical rather than a photochemical intermediate as a limiting factor. We conclude that transmembrane electron transport can utilize products of respiration but that photosynthesis acts independently, perhaps by providing a carbon source which generates reduced substrate.

The role of plasma membrane redox activity in light effects in plants

Stimulations by light of electron transport at the plasma membrane make it possible that redox activity is involved in light-induced signal transduction chains. This is especially true in cases where component(s) of the chain are also located at the plasma membrane. Photosynthetic reactions stimulate transplasma membrane redox activity of mesophyll cells. Activity is measured as a reduction of the nonpermeating redox probe, ferricyanide. The stimulation is due to production of a cytosolic electron donor from a substance(s) transported from the chloroplast. It is unknown whether the stimulation of redox activity is a requirement for other photosynthetically stimulated processes at the plasma membrane, but a reduced intermediate may regulate proton excretion by guard cells. Blue light induces an absorbance change (LIAC) at the plasma membrane whose difference spectrum resembles certainb-type cytochromes. This transport of electrons may be due to absorption of light by a flavoprotein. The LIAC has been implicated as an early step in certain blue light-mediated morphogenic events. Unrelated to photosynthesis, blue light also stimulates electron transport at the plasma membrane to ferricyanide. The relationship between LIAC and transmembrane electron flow has not yet been determined, but blue light-regulated proton excretion and/or growth may depend on this electron flow. No conclusions can be drawn regarding any role for phytochrome because of a paucity of information concerning the effects of red light on redox activity at the plasma membrane.

Effect of iron deficiency on electron transport processes of plasmalemma-enriched vesicles isolated from cotton roots

Abstract To investigate the accelerated transplasmalemma redox activity induced by iron starvation, cotton seedlings ( Gossypium hirsutum L., cv Acala San Jose 2) are grown in nutrient solutions with or without Fe 3+ -ethylenediamine tetraacetic acid (FeEDTA). Vesicles enriched in plasmalemma are then isolated from the roots using a sucrose step gradient or by two-phase partitioning. Iron starvation stimulates by approximately two-fold the transport of electrons from NADH to FeEDTA, ferricyanide or cytochrome c when both donor and acceptor are added at the same face of the membrane. Under these conditions, NADH:duroquinone oxidoreductase activity is unaffected. It is concluded that not all of the redox systems at the plasmalemma are stimulated by iron starvation. Regardless of iron nutrition, Triton X-100 stimulates NADH oxidation of the vesicles when ferricyanide and duroquinone are the electron acceptors, but the detergent inhibits oxidation in the presence of cytochrome c. Transplasmalemma redox activity, which resembles more closely that occurring in vivo, is also detected in the vesicle system by measuring the pH gradient or the membrane potential generated as electrons are transported from ascorbate trapped within the vesicle to exogenous ferricyanide. The pH gradient observed while the membrane is not polarized, is steeper in vesicles from iron-starved roots; however, when the membrane potential is measured, Fe starvation results in its becoming less positive. These results are interpreted as being due to a stimulation by iron starvation of both transplasmalemma electron transport and of proton efflux. Based on the above observations and conclusions, we also suggest that plasmalemma-enriched vesicles provide a satisfactory model system to study aspects of the iron deficiency response.

Effects of peeling on the surface structure of the Avena coleoptile: Implications for hormone research.

Coleoptiles of oats (Avena sativa L.) are often peeled in order to observe hormone-enhanced acidification of the external medium. It is shown by means of the scanning electron microscope that peeling largely removes a single layer of cells, the epidermis with its cuticle. Strips of intact and damaged epidermal cells remain, but most of the newly exposed surface is composed of cortical cells. The cortical face is relatively intact, except that some cells appear punctured and some are broken when a vascular bundle is pulled out with the epidermis. The surface of the cortex is covered by a thin “film” which is partially digested by 2% pectinase. The pectinase pretreatment also inhibits indoleacetic-acid- and fusicoccin-enhanced acidification. Thus, although peeling could be involved in proton extrusion, physical damage to the coleoptile cells per se does not seem to be the major stimulus leading to hormone-enhanced acidification.

The action of ascorbate in vesicular systems

Many effects of ascorbate center on its interactions with membranes from plant and animal cells. These actions can be studied using vesicles produced from phospholipid components (liposomes), by isolating naturally occurring vesicles, or by purifying particular membranes that form vesicles during the extraction process. Liposomes have provided information concerning the anti- and prooxidant properties of ascorbate and about how the water-soluble vitamin can have effects within the phospholipid bilayer. The involvement of ascorbate in transmembrane electron transport has been characterized in vesicles normally found in certain cells, such as, chromaffin granules, synaptosomes, glyoxisomes, peroxisomes, and clathrincoated vesicles. Redox activity using reducing power associated with ascorbate/ascorbate free radical (AFR) has been characterized in some of these vesicles and it appears to be mediated by ab-type cytochrome. Ascorbate also participates in the reduction of iron within clathrin-coated vesicles. Vesicles appearing during purification of plasma membranes have transmembrane electron transport, oxidoreductase activity with ascorbate/AFR as redox agents, and an ascorbate-reducibleb-type cytochrome. It is also possible that ascorbate-related redox activity exists at the tonoplast of plant cells.

Regulation of transplasmalemma electron transport by calcium and light in oat mesophyll cells

The transport of electrons across the plasmalemma from a cytosolic donor to an extracellular acceptor can provide an energy source capable of regulating cellular events. Calcium ions and light, either alone or interacting in some way, control important activities in the cell and evidence exists that both effectors also influence rates of transplasmalemma electron flow.1,2,3,4 We have been attempting to characterize further calcium and light effects on cell surface redox activity. If redox activity and a particular cellular process are both regulated by calcium and/or light, then the possibility exists that redox activity mediates the action of these effectors. An indication that such a relationship may exist with respect to blue light has been obtained in vitro. In this case, the same wavelengths of blue light that lead to absorbance changes characteristic of a reduced b-type cytochrome, are also active in phototropism.5,6