Leaf respiration during early plastidogenesis in light-grown barley seedlings

Oxygen fluxes of protoplasts from different developmental regions of light-grown (16 h day/8 h dark) barley ( Hordeum vulgare L. cv. Patty) seedlings were studied using oxygen electrodes as well as with a more sensitive technique of oxygen exchange measurement, Cartesian-diver microrespirometry. A pronounced increase in dark respiration of protoplasts from the region close to the intercalary meristem was observed, especially below the amyloplast region. In the dark this region also displayed the greatest activity of the alternative (cyanide-insensitive) mitochondrial electron-flow pathway, possibly due to high intracellular sugar levels and suitable cytosolic energy charge engaging the pathway. Etiolated protoplasts from similar regions of dark-grown barley seedlings close to the intercalary meristem displayed a reduced dark respiratory rate which suggests that mitochondrial participation prior to normal plastid development is a partially light-initiated process, probably acting through the agency of phytochrome and a blue light receptor. These results demonstrate that in normal light-grown seedlings, just as in algal and etiolated greening systems, mitochondrial participation is vital for the very early stages of plastid development and may be controlled at least partly through phytochrome action as well as a blue light receptor.     

Inhibition of Respiration in Protoplasts from Meristematic Tissues3

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1986. Inhibitionof respiration in protoplasts from meristematic tissues by abscisicacid in the presence of calcium ions.—J. exp. Bot. 38:498–505. A study was made of the influences of abscisic acid (ABA) andcalcium ions on mitochondrial respiration in protoplasts fromcells close to the basal intercalary meristem of light-grownbarley (Hordeum vulgare L. cv. Patty) seedlings. This respirationwas inhibited by ABA only when calcium ions were present. Thecalcium channel agonist BAY K8644 caused a significant inhibitionof protoplast dark respiration, similar to that observed usingABA and calcium, presumably because it imitated the action ofABA by increasing calcium influx into protoplasts. These resultssuggest that ABA increases the permeability of the plasmamembraneto calcium and that calcium acts as a second messenger to regulatemitochondrial respiratory activity and thus the very early eventsassociated with plastid and meristematic cell development. Key words: Abscisic acid, calcium, meristematic respiration     

Runner Formation in Everbearing Strawberry as Influenced by Growth-Promoting and Inhibiting Substances

The interactive effects of gibberellic acid, abscisic acid,and benzyladenine were studied in relation to runner formationof the everbearing strawberry. In two of three cultivars studied,Ozark Beauty and Superfection, exogenous gibberellic acid stimulatedrunner formation and inhibited flowering. In the Geneva cultivar,a shy runner producer, gibberellic acid application as a foliarspray failed to induce runners. When applied in combinationwith benzyladenine, gibberellic acid greatly stimulated runnerformation Benzyladenine alone had no effect on runnering. Exogenous abscisic acid inhibited petiole length and runnerformation and had no influence on the number of inflorescencesinitiated. Response to exogenous gibberellic acid or abscisicacid varied according to the reproductive or vegetative stagethe plant exhibited at the time of treatment. Activity of endogenous gibberellin-like substances was not substantiallydifferent between flowering and non-flowering field-grown Genevaplants when sampled in long day lengths of August. Endogenousinhibitors were highly active in flowering Geneva plants andrelatively inactive in non-flowering plants The implications of these findings on hormonal balance in thestrawberry are discussed.     

Calcium, Calmodulin and the Control of Respiration in Protoplasts Isolated from Meristematic Tissues8

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1987. Calcium,calmodulin and the control of respiration in protoplasts isolatedfrom meristematic tissues by abscisic acid.—J. exp. Bot.38: 1356–1361. A study was made of the possible involvement of calcium channelsand calmodulin during the calcium-dependent inhibition of mitochondrialrespiration by abscisic acid (ABA) in meristematic protoplastsobtained from light-grown barley (Hordeum vulgare L. cv. Patty)seedlings. The calcium channel blockers lanthanum, verapamiland nifedipine were all found to reduce the Ca²⁺-dependent inhibitionof protoplast respiration by ABA. The ionophore A23187 [GenBank] itselfcaused an inhibition of protoplast respiration, possibly becauseit mimicked the action of ABA by increasing plasmalemma permeabilityto extracellular calcium. By contrast, calmodulin antagoniststrifluoperazine and compound 48/80 both caused a partial decreasein the Ca²⁺-dependent inhibition of protoplast respiration byABA. In contrast to the action of ABA, gibberellic acid markedlyincreased the rates of protoplast respiration but this did notappear to require the presence of extracellular calcium ions.These results support the hypothesis that ABA increases plasmalemmapermeability to extracellular calcium which might then directlyor indirectly act as a second messenger, possibly in conjunctionwith calmodulin, to regulate mitochondrial dark respirationwhich is an important part of early meristematic cell development. Key words: Abscisic acid, calcium, calmodulin, meristematic respiration     

Cartesian-diver microrespirometry: a technique for the accurate measurement of respiratory flux in plant cells

Abstract. A Cartesian-diver microrespirometer system is described which can be used to measure respiratory fluxes of oxygen accurately for cells of higher plants in a liquid phase. This microrespirometry technique has been adapted from protozoological and microfaunal studies to plant physiology. The Cartesian-diver has considerable scope for investigation of oxygen flux in plant cells and has several advantages compared to the oxygen electrode in terms of sensitivity to changing oxygen levels in respiring material. Because the volumes of liquid are small in the Cartesian-divers, diffusional distances arc measured in micrometres and there is no need for stirring to overcome diffusional problems, thus minimizing the risk of mechanical damage to the experimental material. In addition, only very small quantities of experimental material are required for the Cartesian-diver which is invaluable where only limited amounts of tissue or numbers of cells can be obtained. Examples of respiratory oxygen consumption by protoplasts from intercalary meristematic regions of light-grown barley ( Hordeum vulgare L.c.v. Patty) seedlings, in response to abscisic and gibberellic acids, are presented. The advantages and disadvantages of Cartesian-diver microrespirometry compared to oxygen electrodes are also discussed.     

Swelling of Etiolated Oat Protoplasts Induced by Phytochrome, cAMP and Gibberellic Acid: A Kinetic Study

The effects of phytochrome/light and other regulatory agentson the swelling of protoplasts from the primary leaves of etiolatedoat seedlings have been investigated. The protoplasts did notswell in darkness. Red (R) light immediately followed by far-red(FR) or FR light treatment alone for 4 h induced swelling slowly.In comparison, the protoplasts treated with R or FR-R swellmore rapidly and with a shorter lag period. The effect of redlight on the protoplast swelling was photoreversible by FR,suggesting the involvement of endogenous phytochrome. Exogenous gibberellic acid (GA₃) or dibutyryl cAMP (DBcAMP)stimulated the effect of R irradiation on the protoplast swelling.As with R irradiation, these agents were sufficient to causethe swelling of protoplasts in the dark with a shorter lag periodthan those maintained in darkness or a 5 min FR. The combinationof a 5 min R irradiation and GA₃ showed a synergistic effecton the enlargement of protoplast size. On the other hand, theincrease in protoplast size was proportional to the concentrationof DBcAMP with or without a 5 min R irradiation. (Received February 29, 1988; Accepted May 13, 1988)     

Loss of short-day requirement for dormancy breaking of bulbils from gibberellic acid-treated plants in Sedum bulbiferum

Sedum bulbiferum forms bulbils at superterranean nodes under long-day conditions, and the detached bulbils sprout after exposure to short-days [1]. When gibberellic acid was applied to the mother plant at the start of the long-day induction period, the number of bulbils formed increased slightly and these bulbils sprouted on incubation in the dark but not under short days of continuous light. However, when gibberellic acid was applied directly to detached bulbils during incubation, the short-day requirement for sprouting was conserved. Gibberellic acid application to the mother plants enhanced sprouting ability of detached bulbils when incubated under illumination with blue, green or far-red light. However, presence of gibberellic acid during bulbil exposure to light did not induced marked enhancement in sprouting under blue, green of far-red light. Thus, gibberellic acid application to the mother plant modified light and photoperiodic requirements for the sprouting of detached bulbils of S. bulbiferum.     

Photoperiod and light quality effects on rate of dark respiration and on photosynthetic capacity in developing seedlings of Chenopodium rubrum

Development and acclimation of energy transduction were studied in seedlings of Chenopodium rubrum L. ecotype selection 184 (50° 10' N; 105° 35' W) in response to photomorphogenic and photoperiodic treatments. Dark respiration and photosynthetic capacity [nmol O₂ (pair of cotyledons)⁻¹ h⁻¹] were measured with an oxygen electrode. Changes in chloroplast ultrastructure were analyzed concomitantly. After germination, seedlings were grown at constant temperature either in darkness or in continuous light (white, red, far-red and blue) or were subjected to diurnal cycles of light/dark or changes in light quality. Dark respiration was low in far-red light treated seedlings. In red light treated seedlings dark respiration was high and the mean value did not depend on fluence rate or photoperiod. Blue light stimulated transitorily and modulated dark respiration in photoperiodic cycles. Photosynthetic capacity was reduced by far-red light and increased by red light. In response to blue light photosynthetic capacity increased, with indications of a requirement for continuous energy input. Phytochrome and a separate blue light receptor seemed to be involved. In continuous red light a clear cut circadian rhythm of dark respiration was observed. Blue light had a specific effect on chloroplast structure.     

Effects of Blue Light on Cell Elongation and Microtubule Orientation in Dark-Grown Gametophytes of Ceratopteris richardii

Gametophytes of Ceratopteris richardii developed into strap-shapedprothalli in the dark. The prothalli had an apical meristem,a subapical elongation zone and a basal zone where no growthoccurred. Continuous irradiation of blue light inhibited cellgrowth of the elongation zone, although red or far-red lighthad no effect on elongation. Cortical microtubules (MTs) ofsubapical cells reoriented from transverse to oblique or parallelto the growing axis during blue light inhibition. Each regionof the strap-shaped prothallus was irradiated with a micro-beamof blue light. Cell elongation in the subapical region was notinhibited by irradiation at the apical meristem. Irradiationof the subapical region inhibited cell elongation to a similarextent as the whole irradiation control. When a single elongatingcell at one side of the subapical region was irradiated by ablue microbeam, elongation was inhibited in a cell at the irradiateside only, resulting in a bowing prothallus. Cortical MTs reorientedfrom transverse to longitudinal to the cell axis in irradiatedcells only. The results indicate that each cell in the subapicalregion perceives blue light by itself, reorients only its corticalMTs and ceases only its cell elongation, independent of surroundingcells. (Received October 17, 1996; Accepted December 9, 1996)     

Effects of light and growth regulators on leaf unrolling in barley

The influence of light, gibberellic acid, and abscisic acid on unrolling of etiolated barley leaf segments was investigated. Gibberellic acid stimulated unrolling of both illuminated and nonilluminated leaf segments. In contrast, abscisic acid prevented light-stimulated unrolling and abolished the slight unrolling of segments maintained in the dark.     

Control by phytochrome of the synthesis of protein related to senescence in chloroplasts of barley (Hordeum vulgare)

Protein synthesis has been measured in chloroplast isolated from detached leaves of barley ( Hordeum vulgare L. cv. Hassan). The effects of hormone and light treatments of the leaves on chloroplast protein synthesis have been compared with effects on other senescence symptoms. Interruption of the dark with red light retards senescence and increases chloroplast protein synthesis. The effect of red light was reversed by far-red light. Red light did not act additively with kinetin, and it competed with ethylene and abscisic acid, accelerators of senescence, which decreased protein synthesis. In contrast to the interruption of the dark with red light, continuous light decreased chloroplast protein synthesis. It may be concluded effects on chloroplast protein synthesis. The retardation of senescence by continuous light is not necessarily related to P_u Instead, energy provided by photosynthesis may be an important factor.     

Barley leaf unrolling. The proline connection

Unrolling of 1 cm sections, taken between 3 and 4 cm from the apex, of 6-day-old, etiolated barley leaves, was promoted by blue (426 nm) and red (658 nm) light. Accompanying such unrolling was a reduction in the level of the free proline of the tissue. When leaf unrolling was prevented by irradiation with far-red (728 nm) light, or treatment with abscisic acid (ABA) following red light irradiation, the level of proline remained more or less unchanged, at the level of the untreated, dark controls. The proline analogue, azetidine carboxylic acid (AZC) powerfully inhibited the light induced leaf opening, emphasizing the significance of proline-containing, structural and functional proteins in barley leaf unrolling. The inhibition imposed by AZC is partially reversible by added proline.     

Photoreversibility of flower initiation in Lemna perpusilla as affected by the light quality of the main light period

Reversible floral responses of Lemna perpusilla to red and far-redlights appeared only at the beginning of the inductive darkperiod when the 8 hr photoperiod consisted of white or red light.When blue or far-red light was given during the 8 hr photoperiod,the far-red given at the beginning of the dark period scarcelyinhibited flowering; red/far-red reversibility newly appearedat the middle of the dark period. This indicates that the photoregulationsystem in the flowering of L. perpusilla can be converted fromthe Pharbitis type to the Xanthium type by changing the lightquality of the main photoperiod from white or red to blue orto far-red, which is known to be effective for the so-calledhigh-energy photoreaction of photomorphogenesis. (Received July 2, 1975; )     

Light Actions in the Germination of Cocklebur Seeds: IV. DISAPPEARANCE OF RED LIGHT-REQUIREMENT FOR THE GERMINATION OF UPPER SEEDS SUBJECT TO ANOXIA, CHILLING, CYANIDE OR AZIDEPRETREATMENT

Esashi, Y., Fuwa, Nn Kojima, K. and Hase, S. 1986. Light actionsin the germination of cocklebur seeds. IV. Disappearance ofred light-requirement for the germination of upper seeds subjectto anoxia, chilling, cyanide or azide pretreatmenL—J.exp. Bot. 37: 1652–1662. The effects on the germination of positively photoblastic uppercocklebur (X anthium pennsylvanicum Wallr.) seeds by pretreatingwith anoxia, chilling, cyanide or azide, which stimulates theirdark germination, were examined in relation to light actions.Prior to experiments, seeds were pre-soaked at 23 °C inthe dark for 1 or 2 weeks to remove the pre-existing Pfr. Whenthe prctreatment conditions were suboptimal for germinationinduction, the stimulating effects of the pretreatments on germinationduring a subsequent dark period at 23 °C were manifest onlywhen seeds were irradiated with red light before or after thepretreatment Red light promotion was reversed by blue or far-redlight treatment. However, both prc-chilling for 6 d at 8 °Cand prctreatment with 1· 5 mol m ^{– 3} NaN³ for 2d could induce full germination without red light exposure.On the other hand, both pre-exposure to anoxia for 8 d and pretreatmentwith 30 mol m^–3 KCN could induce the dark germinationonly when germination occurred at 33 °C which is known toaugment the ratio of an alternative respiration flux to a cytochromeone. Moreover, the dark germination in response to these inductionswere strongly inhibited by the inhibitors of alternative respiration,propyl gallate and benzohydroxamic acid, applied during a subsequentdark period. It was thus suggested that Pfr has some relationto the operation of two respiration systems of cocklebur seeds,but it is not indispensable to germination of this positivelyphotoblastic seed. Key words: Anoxia, azide, blue light, chilling cyanide, dark germination, far-red light, red light, seed germination, X anthium pennsylvanicum     

Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum

Light stimulates leaf expansion in dicotyledons by increasingapoplastic acidification, cell wall loosening and solute accumulationfor turgor maintenance. Red and blue light enhance growth viadifferent photo-systems, but the cellular location and modesof action of these systems is not known. Here, the effect of red and blue light was studied on transportprocesses in epidermal cells of expanding leaves of the Argenteummutant of Pisum satlvum. Both red and blue light caused extraceiiuiaracidification by isolated epidermal tissue, which was stimulatedby extracellular K⁺ and inhibited by DCCD at 0.1 mol m^–3.Acidification induced by red compared with blue light showeddifferent saturating kinetics in fluence rate-response curves.Under near saturating light conditions the effects of red andblue light were additive. The red light-induced acidificationwas inhibited by far-red light while the blue light-inducedacidification was not. Light caused a hyperpoianzation of themembrane potential in epidermal strips, and stimulated ⁸⁶Rb⁺uptake by epidermal protoplasts. These results show that phytochromeand an additional blue light-photoreceptor function in isolatedepidermal cells to promote proton efflux, hyperpolarization,and cation uptake. Key words: Pisum sativum, light-induced acidification, ion transport, epidermis, photoreceptor     

Germination of Coffee Seeds (Coffea arabica L. cv. Mundo Novo)

The life-span of coffee seeds is extended when seeds are storedwith high moisture content. Germination in darkness is alwayshigher than in the light. Exogenous gibberellic acid and abscisicacid inhibit germination while kinetin reverses this inhibitoryeffect. Low levels of endogenous gibberellin- and abscisic acid-likeand high levels of cytokinin-like substances favour germinationwhile the opposite combination of regulators delays germination.     

Growth and Dormancy in Lunularia cruciata (L.) Dum: VI. GROWTH REGULATION BY DAYLENGTH, BY RED, FAR-RED, AND BLUE LIGHT, AND BY APPLIED GROWTH REGULATORS AND CHELATING AGENTS

Experiments with photoperiods ranging from 2 to 24 h confirmthat 8 h light per day is optimal for Lunularia: there is nogrowth in the dark or in continuous light, which causes therapid onset of dormancy. Short-day cycles intercalated amonga series of continuous light cycles promote growth; in cycleslonger than 24 h very long dark periods are detrimental. Withvery short photoperiods (5 min) red light promotes growth moreeffectively than white light at higher intensity; far-red actsas dark. The growth effects of red and far-red light breaks(3 min) depended on the time of application; red light inhibitedin the middle but promoted at the beginning of the 16-h darkperiod of a short day; far-red light had the opposite effect;in each case red and far-red effects were reversible by theother wavelength. Blue light gave the same response as red includingthe reversibility of far-red effects and vice versa. Surprisingly,significant effects of 5 min red, blue, and far-red irradiationwere also found in the middle of the main high-intensity white-lightperiod, red and blue promoting growth, far-red reducing it;again there was ready reversibility of the effects. Growth promoters of higher plants are generally inhibitory toLunularia or have little effect; among growth retardants TIBA,Phosphon D, and CCC gave a slight promotion of growth. EDTApromoted growth (cell numbers) very significantly while 8-hydroxyquinolinewas initially inhibitory, but had a marked latent promotingeffect when subsequently washed from the thalli.     

Requirement of sodium chloride for the action of gibberellic acid in stimulating hypocotyl elongation of a halophyte, Salicornia herbacea L.

NaCl stimulated hypocotyl elongation of the halophyte Salicorniaherbacea L. grown either in light or dark. Its optimal concentrationwas around 0.1–0.2 M and its promoting effect was muchmore prominent in the dark. Gibberellic acid at 10^–5 Mstimulated hypocotyl elongation in light but not in the dark.Indole-3-acetic acid and kinetin were ineffective in promotinghypocotyl elongation. In light, gibberellic acid and NaCl synergisticallyenhanced hypocotyl elongation when both were given simultaneously.The action of NaCl could be replaced by KCl, but not by mannitol.Osmotic pressure of the epidermis of the Salicornia hypocotylincreased in response to gibberellic acid and/or NaCl treatment.Na⁺ content in the hypocotyl increased with NaCl application.Gibberellic acid and NaCl when given alone increased the extensibilityof the hypocotyl cell wall. Synergistic interaction in increasingthe extensibility was observed between gibberellic acid andNaCl. Stress-relaxation analysis of mechanical properties ofthe hypocotyl wall revealed that gibberellic acid and NaCl actedsynergistically in decreasing minimum relaxation time. Basedon these results, a possible mechanism by which gibberellicacid and NaCl regulate hypocotyl elongation of Salicornia herbaceaL., a typical halophilic plant, is discussed. ¹ Present address: Laboratory of Biology, Tezukayama College,Gakuen Minami, Nara 631, Japan. (Received June 13, 1978; )     

Effects of Far-red Light on the Labelling of Acyl Lipids during the Greening of Barley (Hordeum vulgare)

Etiolated Hordeum vulgare (barley) Plants were greened underwhite light or far-red (> 700 nm) light. Exposure to far-redlight inhibited chlorophyll synthesis (especially chlorophyllb) and the development of photosystem II which were seen whengreening took place under white light. Primary leaves were detachedand the labelling of their acyl lipids from [¹⁴C]acetate wasstudied under white light or far-red light illumination. Greeningwith far-red light caused a reduction in the radiolabellingof polyunsaturated fatty acids and diacylgalactosylglycerol.Total fatty acid labelling rates were unaffected. Phosphatidylethanolamine,which was normally poorly labelled, accounted for up to 15 percent of the total radioactivity in acyl moieties of lipids inleaves greened with far-red light. The results are discussedin connection with the role that acyl lipids may play in normalthylakoid structure and function. Hordeum vulgare, barley, acyl lipids, white light, far-red light, chloroplasts, thylakoids     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.