Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Photophysiology of turion formation and germination inSpirodela polyrhiza

Standardized laboratory techniques for the vegetative growth of the duckweedSpirodela polyrhiza (Lemnaceaé), and for formation as well as germination of their turions were described. Increasing photon fluence rates of blue or red light increased the yield of turions. A specific stimulating effect of blue light was demonstrated under autotrophic but not under mixotrophic conditions. Therefore the spectral composition of light is not important in mixotrophic formation of turions whereas in autotrophic formation light sources with a higher portion of blue light are recommended. Dark-grown (etiolated) turions showed accelerated germination and higher germination percentage in comparison with light-grown turions after induction by a single red light pulse. This difference was overcome in continuous red light by speeding up the germination response of light-grown turions. Use of Petri dishes (8 cm³ nutrient solution) instead of Erlenmeyer flasks (50 cm³ nutrient solution) retarded germination response. Especially for long term experiments the use of Erlenmeyer flasks is recommended. Storage of turions for 72 h at 25 ‡C following at 5 ‡C in darkness after-ripening resulted in a decreased lag phase of the light-induced germination both after induction by a single light pulse and in continuous light. We thank Dr. Halina Gabrys, University of Crakow, Poland for critical discussion.     

Photophysiology of turion germination in Spirodela polyrhiza (L.) Schleiden. X. Role of nitrate in the phytochrome-mediated response

It was shown previously that light-dependent germination of turions of Spirodela polyrhiza (Lemnaceae) is mediated by the photoreceptor phytochrome [Appenroth & Augsten (1990) Photochemistry and Photobiology 52, 61–65]. In the present study, we found that this photoresponse depends on nitrate in the surrounding medium both during after-ripening (under natural conditions occurring in winter) and during germination after light-induction (in spring). The action of nitrate in the germination response is neither related to the induction of nitrate reductase nor to the rate of uptake of ¹⁵NO₃^?. Moreover, two-factor analysis (phytochrome, nitrate) revealed a multiplicative coaction, i.e. independent action of both factors in mediation of germination. The notion that nitrate is a nutritional prerequisite in phytochrome-mediated germination of turions, is supported by the following facts: (1) Nitrate-requirement during germination was strongly increased by nitrate starvation during after-ripening prior to germination. (2) Ammonium could substitute for nitrate. (3) Nitrate uptake by the turions was unaffected by phytochrome and very pronounced even at low concentrations (0.07 mol m^?3) in the medium. With regard to the phytochrome-induced chain of events, it is concluded that nitrate is a prerequisite during a specific developmental phase. Nitrate is not a regulatory element within the chain. In an ecological sense, however, nitrate contents of the aquatic system regulate the germination of turions.     

Light-induced degradation of storage starch in turions of Spirodela polyrhiza depends on nitrate

Light induces both the germination of turions of the duckweed Spirodela polyrhiza and the degradation of the reserve starch stored in the turions. The germination photoresponse requires nitrate, and we show here that nitrate is also needed for the light-induced degradation of the turion starch. Ammonium cannot substitute for nitrate in this regard, and nitrate thus acts specifically as signal to promote starch degradation in the turions. Irradiation with continuous red light leads to starch degradation via auto-phosphorylation of starch-associated glucan, water dikinase (GWD), phosphorylation of the turion starch and enhanced binding of alpha-amylase to starch granules. The present study shows that all of these processes require the presence of nitrate, and that nitrate exerts its effect on starch degradation at a point between the absorption of light by phytochrome and the auto-phosphorylation of the GWD. Nitrate acts to coordinate carbon and nitrogen metabolism in germinating turions: starch will only be broken down when sufficient nitrogen is present to ensure appropriate utilization of the released carbohydrate. These data constitute the first report of control over the initiation of reserve starch degradation by nitrate.     

Germination of Spirodela polyrhiza turions: the role of culture conditions during turion development

The rapidly germinating "old" turions of Spirodela polyrhizawere shown to derive mainly from the slowly germinating "young"turions. This modification to "old" turions could occur evenin isolated "young" turions, and was accelerated by sucrose.It is suggested that this modification is a form of turion senescenceand that turion initiation and maturation are strongly influencedby exogenous carbon and nitrogen sources. (Received November 19, 1979; )     

Low‐molecular weight carbohydrates modulate dormancy and are required for post‐germination growth in turions of Spirodela polyrhiza

The aquatic duckweed Spirodela polyrhiza propagates itself vegetatively by forming turions – bud‐like perennation organs – in the autumn, which spend the winter on the bottom of ponds and then germinate in the following spring and proliferate on the water surface. Newly formed turions usually require a period of cold after‐ripening and light to germinate effectively, but an ample supply of exogenous sugar can lead to germination even in the dark and independent of after‐ripening. The results of the present study indicate that the availability of readily metabolised carbohydrates is a determining factor for turion germination. Freshly harvested turions do not contain soluble, low‐molecular weight carbohydrates at a level sufficient to allow germination to take place, but after‐ripened turions do. Augmentation of the soluble carbohydrate content during after‐ripening derives from gradual breakdown of reserve starch of the turions. The long time required for any germination to be observed in turions incubated in darkness and the limited frequency of germination in the dark (about 50% of turion population), even with an ample external sugar, supply emphasise that both after‐ripening and light are essential for ensuring rapid germination and subsequent frond proliferation at an ecologically appropriate time. The carbohydrate supply required for rapid proliferation of the fronds produced at germination is provided by the rapid light‐induced breakdown of turion reserve starch.     

Turion formation and behaviour inSpirodela polyrhiza at two levels of phosphate supply

The clone SJ ofSpirodela polyrhiza (L.) Schleiden forms turions under various nutritive conditions. As compared to 1500 μmol 1⁻¹ phosphate (P+), growth and frond yield of mixotropic cultures decreased, when 60 μol 1⁻¹ phosphate (P-) were available. By contrast, P-conditions increased the number, individual size, dry matter content, and total turion yield (mg turions per ml of the nutrient medium) of P-turions as compared to P+ ones. Germination behaviour of P-turions is characterized by fairly low zero levels in the controls, and by low heterogeneity in individual size as well as in the response patterns concerning the influence of light and/or phytoactive substances. P-turions from youngSpirodela cultures are extremely dormant. However, they undergo an after-ripening process if kept inside ageing cultures.     

TURION FORMATION AND GERMINATION IN SPIRODELA POLYRHIZA

Three clones of Spirodela polyrhiza L. (Schleid.) formed dormant bodies called turions. A clone from Puerto Rico did not form turions under all conditions tried. In those clones producing turions, formation was stimulated by the addition of sucrose (10–50 mM) to the nutrient solution. Increased levels of Ca(NO₃)₂ plus sucrose stimulated turion production. In the absence of NO₃^–, Ca⁺⁺ was more effective than K⁺ in stimulating turion formation. Turion buoyancy was not light dependent, nor was it promoted by sucrose. Normal turions required light for germination, whereas sucrose-induced turions germinated in the dark. Dark germination was not promoted by either Ca⁺⁺ or K⁺. Sucrose stimulation of turion formation and subsequent promotion of dark germination was attributed to metabolic rather than osmotic effects. One hundred mM sucrose concentrations inhibited turion buoyancy and germination. Turions formed one primary abscission layer which separated them from the stolon and the mother frond. Subepidermal idioblasts appeared to seal the stolon stump after separation.     

Photoinduction of Spore Germination in a Fern, Mohria caffrorum

Irradiation of spores of the fern Mohria caffrorum Sw. witheither red light (67.4 µW cm^–2) or far-red light(63.2 µW cm^–2) for a period of 24 h induced maximumlevels of germination. Brief irradiations with blue light (127.6µW cm^–2) administered before or after photoinductioncompletely nullified the effects of red or far-red light; however,with prolonged exposure to blue light, germination levels roseto near maximum. The similar effects of red and far-red lightin promoting spore germination makes the involvement of phytochromein this process questionable. Based on energy requirements,the promotive and inhibitory phases of blue light appear toinvolve independent modes of action. Mohria caffrorum, ferns, spore germination, photoinduction, phytochrome     

The Interactive Effects of Phytochrome, Nitrate and Thiourea on the Germination Response to Alternating Temperatures in Seeds of Ranunculus sceleratus L.: A Quantal Approach

Probert, R. J., Gajjar, K. H. and Haslam, I. K. 1987. The interactiveeffects of phytochrome, nitrate and thiourea on the germinationresponse to alternating temperatures in seeds of Ranunculussceleratus L.: A quantal approach.—J. exp. Bot. 38: 1012–1025. The interactive effects of phytochrome, potassium nitrate andthiourea on the germination response to alternating temperaturesin achenes (seeds) of Ranunculus sceleratus L. were studied.Using thermogradient bars, high levels of germination were recordedover a broad range of alternating temperatures providing seedsreceived daily irradiations. Reduced germination in temperaturecycles with a relatively long warm phase was related to thelevel of the active form of phytochrome (Pfr). Dose-responseexperiments to red light (R) and temperature shifts showed thatthe actions of Pfr and alternating temperatures were interdependent.Maximum germination was recorded when intermittent pulses ofR were combined with daily 4 h temperature shifts from 16°Cto 26°C. Whilst probit analysis showed that potassium nitrateand thiourea both increased population sensitivity to temperatureshifts, thiourea was a more potent stimulant. Although the effectof both chemicals was dependent on phytochrome photo-equilibriumthe threshold level of Pfr required for thiourea action wasclearly much lower than that required for nitrate action. Thioureapotentiated a response to daily temperature shifts even whenPfr was at a low, normally inhibitory level. These results indicatedifferent mechanisms of action for potassium nitrate and thioureain relation to phytochrome controlled seed germination. Key words: Phytochrome, nitrate, thiourea, alternating temperatures, germination     

Heterogeneity of dormancy in the turions of Spirodela polyrrhiza

Spirodela polyrrhiza exhibits at least two different types ofdormancy in turions induced by nitrogen deficiency, i.e., Y(young)- and O (old)-type turions produced at early and latestages in the formation process, respectively. On the otherhand, turions induced by relatively low temperature are mainlythe Y type. O-type turions germinate rapidly under darkness with the additionof NO₃-. However, under semi-anaerobic and anaerobic conditionsthey germinate only when exposed to light in the presence ofNO₃-. Y-type turions germinate in air after a definite dormant periodonly under light and in the presence of NO₃-. The dormancy isbroken by light regardless of the presence of NO₃-, althoughthe germination phase requires both. This dormancy is also brokenwhen a definite period of darkness is given in the absence ofNO₃-, and the germination proceeds in the presence of NO₃- withor without light. Thus, an alternative process exists for theY type in which the dormancy is released by either light ordarkness. (Received October 3, 1978; )     

Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome

The appearance of glutamine synthetase (GS. EC 6.3.1.2) in response to light and nitrogen (NO ₃ ^- , NH ₄ ⁺ ) was studied in the organs (roots, hypocotyl, cotyledonary whorl) of the Scots pine (Pinus sylvestris L.) seedling. Although GS activity was found to be mainly (> 80%) located in the whorl where it increased strongly in response to light, a significant GS synthesis was also detected in dark-grown seedlings. Anion-exchange chromatography was used to resolve two GS isoforms which appeared to be regulated differentially in the cotyledonary whorls. The isoform (presumably plastidic GS2) which eluted from the column at 90 mM KCl increased drastically in response to light. The other isoform (presumably cytosolic GS1), which eluted at 200 mM KCl, was not stimulated by light but tended to disappear during the experimental period (4 to 12 d after sowing). Immunoblotting of pine extract yielded a prominent band with a molecular weight of 43 kDa. The linear correlation between GS activity and immunodetectable GS protein could be extrapolated through zero, showing that any increase of GS2 activity is to be attributed to the de-novo synthesis of GS protein. Gelfiltration chromatography yielded a molecular mass for the GS holoenzyme of 340 kDa, a value which supports an octameric quarternary structure as previously suggested for angiosperms. While supplying seedlings with 10 mM NO ₃ ^- stimulated GS synthesis in the whorl by 12%, 10 mM NH ₄ ⁺ caused an incipient ammonium toxicity. Experiments using dischromatic light (simultaneous treatment with two light beams to vary the level of the physiologically active form of phytochrome, Pfr, in blue light) revealed that synthesis of GS2 was controlled by light in the same way as previously shown for ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1). Up to 10 d after sowing the strong light effect could be attributed to phytochrome action whereas between 10 and 12 d after sowing phytochrome control of GS-synthesis failed if no blue/ultraviolet-A light was provided. The data show that blue light is required to maintain responsiveness of GS2 synthesis to phytochrome. Both enzymes, GS2 as well as Fd-GOGAT, appear to be regulated coordinately to meet the demands of ammonium assimilation.Abbreviations and Symbols B blue light - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1); - GS glutamine synthetase (EC 6.3.1.2) - R red light - RG9 long-wavelength far-red light defined by the properties of Schott glass filter RG9 - =Pfr/Ptot far-red absorbing form of phytochrome/total phytochrome, wavelength-dependent photoequilibrium of the phytochrome system Research supported by Deutsche Forschungsgemeinschaft (SFB 46 and Schwerpunkt Physiologie der Bäume). We thank J.M. Penther, (Institut für Biologie II, Freiburg, FRG) for his advice on the chromatographic techniques.     

Phytochrome-induced transmissible signal elicits germination response in turions of Spirodela polyrhiza

The role of cell competence, including the spatiotemporal aspect of phytochrome-induced long-distance signal transmission, was investigated in turions of Spirodela polyrhiza (L.) Schleiden. Irradiation of the dorsal surface of the turions triggered a significant germination response, while identical treatment of the ventral surface was less effective. Red-light (R) microbeam irradiation of a subregion (ca. 1 μm²) of the dorsal surface could induce the germination response. Therefore, photoactivation of phytochrome in a single cell or few cells is sufficient to trigger the photomorphogenetic response. The ultimate response occurs at the proximal end of the turion by way of growth and emergence of the frond primordia about 1.3 mm away from the microbeam-irradiated distal cell(s). This photoinduction was reversible by a pulse of far-red light (FR) given less than 24 h after R microbeam irradiation. Microsurgical separation of distal (irradiated) and proximal (primordium-bearing) halves of the turions following microbeam irradiation further revealed that the light-induced transmissible signal can be intercepted and that it required more than 48 h to traverse one half distance of the turions. Based on the kinetics of the signal transmission, the possible involvement of light scattering, light piping, or transfer of electrophysiological signals can be excluded. Taken together, the results indicate that a transmissible signal is generated by the irradiated cell(s) and propagated across to the non-irradiated cells, leading to induction of the photomorphogenetic response.     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light     

STIMULATION OF NITRATE NET UPTAKE AND REDUCTION BY RED AND BLUE LIGHT AND REVERSION BY FAR-RED LIGHT IN THE GREEN ALGA ULVA RIGIDA1

The steady-state levels of nitrate, nitrite, and ammonium were estimated in the green alga Ulva rigida C. Agardh in darkness after addition of 0.5 mM KNO₃ and irradiation with red (R) and blue (B) light pulses of different duration (5 and 30 min). The net uptake of nitrate was very rapid. Seventy-five percent of the nitrate added was consumed after 60 min in darkness. Although uptake was stable after R or B, efflux of nitrate occurred within 3 h in the dark control and when R or B were followed by far-red (FR) irradiation. The internal nitrate concentration after 3 h in darkness was similar after R and B light pulses; however, the intracellular ammonium was higher after R than after B. The intracellular nitrate and ammonium decreased when FR tight pulses were applied immediately after R or B. Thus, the involvement of phytochrome in the transport of nitrate and ammonium is proposed. Nitrate reductase activity, measured by the in situ method, was increased by both R and B light pulses. The effect was partially reversed by FR light. Nitrate reductase activity was higher after 5 min of R light than after 5 min of B. However, after 30-min light pulses, the relative increase in activity was reversed for R and B. We propose that phytochrome and a blue-light photoreceptor are involved in regulation of nitrogen metabolism. Nitrate uptake and reduction correlates with previously detected light-regulated accumulation of protein in Ulva rigida under the same experimental conditions.     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)