Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Uptake of Iodide by Excised Bean Roots

Mesophyll cells of leaf slices of bean (Phaseolus vulgaris L.) absorb six to ten times more K⁺ than Rb⁺ from 0.1 mM single chlorides of these cations. Absorption of ⁴²K⁺ from 0.1 mM⁴²KCl is much more inhibited by low concentrations of Rb₂SO₄ than by K₂SO₄. The isotherm for K⁺ absorption is biphasic in the range 0.1–1.1 mM, and K⁺ is more effective than Rb⁺ in causing transition from phase 1 to phase 2.     

Role of Membrane-bound, Fixed-charge Changes in Phytochrome-mediated Mung Bean Root Tip Adherence Phenomenon

The movement of cells and cell fragments in an electric field provided a means for determining the nature of cellular surface charges. We found that changes in ionic strength and particularly changes in Ca²⁺ and H⁺ in the bathing medium cause changes in the surface charges on the root cap cells in the absence of red light. Red light-induced charge changes are demonstrable only on root cap cells and are reversible with far red light. By osmotically separating the membrane from the wall, we demonstrated that both light-induced and ionically mediated charge changes are associated with the cell membrane and not the cell wall.     

Rapid Phytochrome-mediated Changes in Adenosine 5'-Triphosphate Content of Etiolated Bean Buds

This study was designed to determine the effects of red and far red irradiation on ATP metabolism in etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney). Compared to dark controls, red irradiated buds show an initial decline in ATP content at 15 seconds following a 5-minute irradiation. ATP content then rapidly rises to a peak at 1 minute, and then slowly returns to the baseline. The 1-minute promotion of ATP content is red/far red reversible. Acetylcholine does not appear to mimic red light in this system; it causes a marked decrease in ATP content.     

Acetylcholine and Synthesis of Ethylene in Etiolated Bean Tissues

Acetylcholine chloride inhibited ethylene production in etiolated bean tissues and prevented to some extent the IAA-promoted opening of the bean hypocotyl hooks. The results were interpreted on the basis of acetylcholine mimicking the effects of red light on auxin content and ethylene production.     

Evidence for G-Protein Regulation of Inward K+ Channel Current in Guard Cells of Fava Bean

Recent reports have shown that GTP-binding proteins (G-proteins) are present in plants but have given limited indication as to their site of action. G-proteins in animal cells transduce extracellular signals into intracellular or membrane-mediated events, including the regulation of ion channels. Using whole-cell patch clamp, we provide evidence that a G-protein in guard cells of fava bean regulates the magnitude (and not the kinetics) of inward current through K+-selective ion channels in the plasma membrane. GDP[beta]S (100 to 500 [mu]M) increases inward K+ current, whereas GTP[gamma]S (500 [mu]M) has the opposite effect. The control nucleotides ADP[beta]S and ATP[gamma]S (500 [mu]M) do not affect K+ current. Reduction of inward current by GTP[gamma]S is eliminated in the presence of the Ca2+ chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N[prime],N[prime],-tetraacetic acid) (5 mM). When applied intracellularly, the G-protein regulators, cholera toxin and pertussis toxin, both decrease inward K+ current. The entry of K+ (and anions) into guard cells increases their turgor, opening stomatal pores in the leaf epidermis that allow gas exchange with the environment. Our data suggest the involvement of a G-protein in the inhibition of K+ uptake and stomatal opening. Changes in stomatal aperture, vital to both photosynthesis and plant water status, reflect guard-cell responsiveness to a variety of known environmental signals. The results presented here indicate that, in plants as well as animals, ion channel regulation by environmental stimuli may be mediated by G-proteins.     

Evidence for Repair Processes in the Invigoration of Seeds by Hydration

Seeds with low vigour as a result of ageing showed a large improvementin seed quality following a 2 h soak in water, reflected inan increased rate of germination, higher emergence in soil,and the retention of high germination after 24 h rapid ageingin a controlled deterioration vigour test. Similar, but smallerimprovements were seen after a priming treatment using polyethyleneglycol. Seeds dried to their initial moisture content aftertreatment retained these beneficial effects. The treatmentshad little effect on high vigour seed. These findings suggestthat seed invigoration by hydration treatments results fromthe repair of previously sustained deterioration. Brussels sprouts, Brassica oleracea var. gemmifera, priming, repair, seed moisture content, ageing     

Regulation of Mitosis in Roots by their Caps

WHEN the meristematic cells of root tips are damaged by surgery, X-rays or various chemical treatments, the cells of the quiescent centre (Fig. 1) are stimulated into mitosis. These cells normally have an average rate of mitosis about one-tenth of that of their neighbours more than half of them are not in mitotic cycles and, of those that are, some have a “fast” cycle three or four times as long as those of their neighbours¹. After X-irradiation there is an immediate response in the quiescent centre that suggests that the balance in cell proliferation between different regions of the meristem is not a simple competition effect. One stimulatory action is the removal of the root cap² and I have now found that the cap exerts a complex effect on mitosis in the rest of the meristem.     

Rapid Respiratory Changes Due to Red Light or Acetylcholine during the Early Events of Phytochrome-mediated Photomorphogenesis

Two millimeter long secondary root tips of etiolated mung bean (Phaseolus aureus) plants were given 4 minute consecutive treatments of darkness, red light, far red light, and acetylcholine during darkness. We studied the effects of these treatments on exogenous (H⁺) changes, ATP utilization, O₂ uptake, P₁ levels, and ATPase activity. Red light and acetylcholine increased the level of P₁, O₂ uptake, and exogenous H⁺, but decreased ATP concentrations. Darkness and far red light caused the amount of ATP to increase and decreased the O₂ uptake and P₁ level. O₂ uptake of both excised root tips and isolated mitochondria was promoted by acetylcholine levels of the same order of magnitude that promoted the other photomimetic phenomena. ADP-O ratios indicated that acetylcholine did not cause an appreciable decrease in ATP synthesis. The total ATPase activity remained constant throughout all treatments. Ouabain caused no adhesion to negatively charged glass in the dark, while the inhibitors valinomycin, atractyloside, digitoxin, gramicidin, and oligomycin caused immediate adhesion. All of the inhibitors prevented release from the glass. In red light ouabain increased adhesion, whereas the other inhibitors caused caused immediate and complete adhesion.     

Specificity of Muscarinic Acetylcholine Receptor Regulation by Receptor Activity

Abstract— Regulation of muscarinic acetylcholine receptor concentration by receptor activity in neuron-like NG108-15 hybrid cells is a highly specific process. Receptor levels, monitored by binding of [³H]quinuclidinyl benzilate ([³H]QNB), decreased 50-75% following 24-h incubation of cells with muscarinic agonists, but none of the following cellular processes was altered by this chronic receptor stimulation: (1) glycolytic energy metabolism, measured by [³H]deoxy- d -glucose ([³H]DG) uptake and retention; (2) rate of cell division; (3) transport, measured by [³H]valine and [³H]uridine uptake; (4) RNA biosynthesis, measured by [³H]uridine incorporation; (5) protein biosynthesis, measured by [³H]valine and [³⁵S]methionine incorporation into total protein and into protein fractions obtained by polyacrylamide gel electrophoresis. In contrast, chronic stimulation did cause a threefold decrease in the capacity of carbachol to stimulate phosphatidylinositol (PI) turnover, a receptor-mediated response. In addition to cholinomimetics, the neuroeffector adenosine (1 m m for 24 h) also caused a decrease in [³H]QNB binding levels, but chronic stimulation of α -adrenergic, opiate, prostaglandin E₁, and prostaglandin F_2α receptors found on NG108-15 cells caused no changes. The data indicate that loss of muscarinic receptors caused by receptor stimulation is not a consequence of fundamental changes evoked in overall cellular physiology but reflects a specific regulation of cholinoceptive cell responsiveness.     

Evidence for the Active and Passive Chemotropisms in Roots

Changes in the elongation of root cells during the negative (away from the salt) or positive (towards the salt) chemotropic bending of roots induced by the unilateral application of agar blocks (1 mm³) with 10^–3and 10^–2M Cd(NO₃)₂solutions to the meristem zone of the root were studied. The root bending was not accompanied by differential changes in the number of cells that elongated during the 3-h period of chemical stimulation. The bending was only due to differential changes in the cell elongation rates. In most chemically stimulated roots, both concentrations of Cd(NO₃)₂solutions inhibited cell elongation at the stimulated and nonstimulated sides. Cell elongation was inhibited by 10^–2M Cd(NO₃)₂mainly on the stimulated side of the roots, hence, the roots bent towards the salt. On the contrary, 10^–3M Cd(NO₃)₂inhibited cell elongation mainly at the nonstimulated side of the roots. As a result, the roots bent away from the salt, i.e., in the direction opposite to that expected in the case of the direct inhibition of cell growth by Cd(NO₃)₂. It is concluded that the root chemotropisms induced by the above two Cd(NO₃)₂concentrations are, correspondingly, of a passive or active nature.