Clonal Differences in the Formation of Turions are Independent of the Specific Turion-inducing Signal in Spirodela polyrhiza (Great Duckweed)

Abstract: Turion (survival organ) formation in Spirodela polyrhiza includes a switch in the programming of the primordia from the formation of vegetative fronds toward resting turions. The specific turion yield (SY; number of turions formed by one frond) is used to evaluate the effect of three turion-inducing signals: low phosphate concentration (depleted due to frond growth), low temperature (15 °C) and exogenously applied abscisic acid (1 μM). The formation of turions was observed in the presence of any of the turion-inducing factors in all three clones of S. polyrhiza investigated (clones 9256 from Finland, SJ from Germany and SC from Cuba). The clone SC showed no specific induction by low temperature or phosphate limitation in one nutrient medium. Regardless of the specific signal applied, the SYs were highest in clone 9256 and lowest in clone SC, demonstrating signal-independent clonal differences. Clonal differences are therefore located in the developmental-specific common phase of the transduction chains leading to turion formation. We intend to use clonal differences in the molecular analysis of turion formation, e.g., by cDNA-based amplified fragment length polymorphism, to distinguish signal-specific and developmental-specific gene expression. In contrast, the total turion yield is useful in an ecological context to evaluate the number of turions available to support the survival of a population of plants but gives little information about the physiological process.     

Co‐action of temperature and phosphate in inducing turion formation in Spirodela polyrhiza (Great duckweed)*

Increased phosphate concentration, higher temperature and addition of glucose all increased the number of fronds and turions of the duckweed Spirodela polyrhiza formed under in vitro conditions. Increasing the number of turions by increasing the plant biomass does not mean that the developmental process (switch of the programme of the primordia from vegetative fronds toward resting turions) has been specifically influenced. The specific turion yield (STY; number of turions formed by one frond) and the time of onset of turion formation have been used as more specific measures of turion induction. At more than 30 µm initial phosphate the STY was increased by lower temperature (15 °C) and became independent of the phosphate concentration. Between 10 and 30 µm and at higher temperatures (25 °C) the STY was increased by lower phosphate levels. The stimulatory effect of lower temperature was more pronounced than that of lower phosphate concentrations. Decreased phosphate concentration highly accelerated the formation of the first turions. The influence of low temperature was small at lower phosphate concentration but became dominant at higher concentrations (especially in autotrophic cultures). Low phosphate levels (e.g. 10 µm ) and low temperatures (e.g. 15 °C) both represent specific turion‐inducing factors having significant interactive effects. In S. polyrhiza, these signals may replace the interactive effects of photoperiods and low temperature known from other hydrophytes in turion induction under natural conditions.     

Dormancy, Turion Formation, and Germination by Different Clones of Spirodela polyrrhiza

Some clones of Spirodela polyrrhiza form dormant bodies called turions which require several weeks of chilling treatment before they proceed to renew growth and develop into vegetative fronds. The individual fronds of Spirodela are less than 5 mm long and can be grown aseptically in liquid culture. Turion formation and germination can serve as a bioassay for the various compounds involved in dormancy development.

Turion formation can be induced by manipulation of light intensity during the day, photoperiod, night temperature, day temperature, and concentration of nitrate in the culture medium. Different clones of Spirodela from northeastern United States, Puerto Rico, and Argentina had different requirements for turion formation. The clones from Argentina and Puerto Rico did not form turions under any of the experimental conditions imposed. Turions of some clones required chilling treatments for renewed vegetative growth while others did not. Both gibberellic acid and long photoperiods were required to bypass the chilling requirements of some clones, but not others.

     

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.     

Environmental and hormonal control of turion formation in Myriophyllum verticillatum

The turions of Myriophyllum verticillatum, an aquatic vascularplant, develop in the fall and function in propagation and dispersalas well as in over-wintering. Experiments with controlled evnironmentsindicate that both temperature and photoperiod regulate turionformation. Turions can be induced at 15°C or lower, butnot at 20°C. At 15°C, turions form in both 8- and 12-hrdays, but not in 16-hr days. Plants collected in early springdo not form turions readily in response to short days unlesspreviously exposed to long days; thus, turion formation is along-day-short-day response. This combination of photoperiodand temperature requirements probably prevents turion developmentin early spring when the temperature and photoperiod are similarto those in the fall. Treatment of plants with ABA (10^–5M) enhances turion development under marginally inductive conditions(12-hr days at 15°C) but cannot induce it under long days.On the other hand, the cytokinin benzyladenine (10^–5 M)blocks turion formation. GA3 (10^–5 M) and AMO-1618 (10^–5M) exert only small qualitative effects on turion development,while IAA (10^–5 M) retards it. During turion development,the level of ABAlike activity and of one or two unidentifiedinhibitors increases. Cytokinin activity decreases at the startof turion formation, increases during development, then decreasesat abscission. Thus two lines of evidence suggest that a decreasein cytokinin activity and an increase in acidic inhibitor activityplay important roles in turion induction. ¹Present address: Biological Station, University of Michigan,Ann Arbor, Michigan 48109, U. S. A. (Received December 1, 1975; )     

Photophysiology of turion germination inSpirodela polyrhiza (L.)Schleiden. The cause of germination inhibition by overcrowding

Red-light-induced (via phytochrome) germination decreased with increasing numbers of turions per germination flask (overcrowding). Three hypotheses concerning the mechanism of this germination inhibition were tested, related to abscisic acid, ethylene, and oxygen deficiency: (i) Although abscisic acid is a powerful inhibitor of turion germination it had to be excluded as a cause, because abscisic acid was not secreted from turions into the nutrient solution, (ii) Ethylene (ethrel) strongly inhibited growth of newly formed sprouts, but germination response itself was not inhibited, (iii) Germination inhibition did not appear if short light pulses were substituted by continuous irradiation. It reappeared in the presence of the photosynthesis inhibitor 3-(3, 4-dichlorophenyl)-l, 1-dimethylurea, but it was not observed in aerated nutrient solutions, or when Petri dishes instead of Erlenmeyer flasks were used. Decreased oxygen concentrations in the nutrient solution were produced by turion respiration. Consequently, anaerobiosis within the nutrient solution caused by turion respiration was the reason for germination inhibition by overcrowding.     

Turion formation in Spirodela polyrhiza: The environmental signals that induce the developmental process in nature

The formation of turions of Spirodela polyrhiza is induced by a large number of environmental signals, already investigated under laboratory conditions. To get more close‐to‐nature experimental conditions, chemical composition and temperature of the water were measured during the growing season in 2002 and 2003 in a pond near Jena (50°52′N, 11°42′E). Whereas the concentrations of nitrate and sulphate (both in the millimolar range) remained fairly constant that of phosphate decreased from approximately 13 µM at the beginning of the season to 2 µM at the time of onset of turion formation (17 August in 2002, 21 July 2003). This concentration was used in experiments under controlled conditions together with the other outdoor data (day temperature, lower night temperature and photoperiod) in subsequent experiments to investigate their role in the induction of turion formation. The concentration of the nutrient media were kept constant. The following conclusions were drawn. (1) Low phosphate concentration appears to be the decisive factor in inducing turion formation. Growing fronds take up phosphate, and turion formation is then induced towards the end of the season. (2) Lower temperatures during the day (18 vs 25°C) and especially during the night (18 vs 15°C) evidently enhance the effect of the turion‐inducing factor phosphate by increasing the yield. (3) At much higher anthropogenic phosphate concentrations low temperature takes over the function of inducing turion formation. (4) Whereas much lower concentrations act directly to induce the formation of turions regardless of the season.     

Cell wall polysaccharidase activities and growth processes: a possible relationship

The turions of Potamogeton crispus L. develop in early summer and function in propagation and dispersal. Under natural conditions during longday periods, an average minimum air temperature of more than 12°C was found to be important for turion formation. Experiments with controlled environments indicate that both temperature and photoperiod regulate turion formation. Turions can be induced at 13°C or above but not at 8 or 10°C. At a temperature range of 13–24°C turions form in both 12- and 16-h days, but not in 8-h days. By increasing temperature from 24 to 30 or 35°C turions can be induced under 8-h days. Light intensity was found to be important in the formation of turions.     

The clonal dependence of turion formation in the duckweed Spirodela polyrhiza—an ecogeographical approach

Formation of turions, the vegetative perennation organs, plays an important role in the survival strategy of Spirodela polyrhiza (L.) Schleiden. Turion formation [quantified as number of turions formed per frond; specific turion yield (SY)] was investigated in 27 clones collected from a wide geographical range. The Pearson correlation was tested with (1) duration of growing season (monthly average temperature of ≥10°C), (2) relative growth rate of the fronds, (3) longitude and latitude, and (4) several climatic parameters, in all possible single and multiple regressions. All single coefficients of determination were below 0.10. The highest correlation (R² = 0.61; adjusted for the number of explaining variables 0.54) was found in a multiple linear regression with the following five parameters: average temperatures over the year and during the growing season, duration of the growing season and precipitation over the year and during the growth period. All these parameters were shown to have significant contributions. This equation was used successfully to predict the SY of five newly isolated clones. Finally, on the basis of all 32 clones the following conclusions were drawn: The mean annual temperature has the highest impact. It is suggested that lower temperatures decrease the survival rate of turions and that adaptation refers to increasing SY. The different levels of SY in the clones (ranging from SY = 0.22 to 5.9) were detected even after several years of in vitro cultivation. It is therefore assumed that these adaptations to the climatic conditions are genetically determined.     

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     

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.     

Changes in vectors of endogenously generated ion currents in light-induced germination of turions of Spirodela polyrrhiza

Nondormant turions of Spirodela polyrhiza (L.) Schleiden were utilized to investigate endogenous ion currents in light-induced germination and early growing processes of higher plants. A small outward current was detected at the ventral side of the turions near the pocket containing the most developed sprout primordium. After a light pulse, the direction of the endogenous current changed from outward to inward. These ion currents are most likely conditioned by unspecific diffusion of cations (probably H+) inside the cell. Three-day-old sprouts of Spirodela showed the highest inward current near the sprout base which decreases toward its edge. Newly formed sprouts demonstrated a strong gravity effect (bending), which was preceded by a lowering of the Z-component of vectors close to the sprout base after a change of the turion fixation.     

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.     

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68–73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs. Handling editor: S. M. Thomaz     

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.     

Twenty-four-hour profiles of serum leptin in siberian and golden hamsters: photoperiodic and diurnal variations

Serum leptin concentrations were obtained from male Siberian hamsters (Phodopus sungorus) and golden hamsters (a.k.a. Syrian, Mesocricetus auratus) housed on long [light:dark (LD) 16:8] and short (LD 6:18) photoperiods for 10-11 weeks. Blood samples were collected at 45-min intervals for 24 h from individual animals using an in-dwelling atrial catheter. In Siberian hamsters, exposure to short photoperiods as compared to long photoperiods reduced body weight (32.5 +/- 1.5 vs 47.7 +/- 1.1 g) and leptin (24-h mean: 5.3 +/- 0.4 ng/ml vs 18.6 +/- 2.1 ng/ml). Although photoperiod influenced the temporal distribution of leptin in golden hamsters, the main effect of photoperiod on leptin levels in golden hamsters did not reach significance (24-h mean: 7.1 +/- 1.0 ng/ml vs 5.1 +/- 0.8 ng/ml.). Body weights of golden hamsters did not vary significantly following exposure to short photoperiod for 11 weeks (178.3 +/- 3.6 g in LD 6:18 vs 177.8 +/- 7.3 g in LD 16:8). There was no nocturnal increase in serum leptin in either species. Marked interindividual differences were apparent in individual leptin profiles. Periodogram analysis revealed that only a few animals exhibited 24-h periodicities; the presence of a significant 24-h periodicity was more common in hamsters exposed to short days. Photoperiod-associated differences in the 24-hour profile of leptin secretion may be the result of photoperiod-associated changes in feeding behavior or metabolism. A full understanding of the regulation of leptin secretion in multiple time domains may enhance our understanding of the function of leptin.     

Influence of salinity and high temperature on turion formation in the duckweed Spirodela polyrhiza

The influence of NaCl (salinity; 0-5 ‰) and higher temperature (heat stress; 32 °C) on yield of turion formation has been tested in the duckweed Spirodela polyrhiza for the first time. Turion formation was more sensitive to both stressors than the growth of the vegetative fronds: (1) the concentration of NaCl which produces half-maximal inhibition was lower for turion formation than for growth by the factor of five. (2) At 32 °C turion formation was completely blocked whereas growth rates decreased by only 20% as compared with 28 °C.     

The Physiological Role of Abscisic Acid in Eliciting Turion Morphogenesis

The exogenous application of hormones has led to their implication in a number of processes within the plant. However, proof of their function in vivo depends on quantitative data demonstrating that the exogenous concentration used to elicit a response leads to tissue hormone levels within the physiological range. Such proof is often lacking in many investigations. We are using abscisic acid (ABA)-induced turion formation in Spirodela polyrrhiza L. to investigate the mechanism by which a hormone can trigger a morphogenic switch. In this paper, we demonstrate that the exogenous concentration of ABA used to induce turions leads to tissue concentrations of ABA within the physiological range, as quantified by both enzyme-linked immunosorbent assay and high-performance liquid chromatography/gas chromatography-electron capture detection analysis. These results are consistent with ABA having a physiological role in turion formation, and they provide an estimate of the changes in endogenous ABA concentration required if environmental effectors of turion formation (e.g. nitrate deficiency, cold) act via an increased level of ABA. In addition, we show that the (+)- and (-)-enantiomers of ABA are equally effective in inducing turions. Moreover, comparison of the ABA; levels attained after treatment with (+)-, (-)-, and ([plus or minus])-ABA and their effect on turion induction and comparison of the effectiveness of ABA on turion induction under different pH regimes suggest that ABA most likely interacts with a plasmalemma-located receptor system to induce turion formation.     

Life cycle and growth ofPotamogeton crispus L. in a shallow pond,ojaga-ike

The life cycle and growth ofPotamogeton crispus L. were studied in a shallow pond, Ojaga-ike. With respect to the shoot elongation and seed and turion formations, the life cycle of this plant in the pond could be divided into following five stages: germination, inactive growth, active growth, reproductive and dormant stages. It was suggested that the plant showed these successive stages depending mainly upon water temperature. The turions germinated on the bottom in autumn when the water temperature fell below ca. 20 C. The plant showed hardly any growth during winter (December—early March) when the temperature was below 10 C. In the spring when the bottom water temperature rose to above 10 C (mid-March), the plant started to grow again and the shoot elongated rapidly at the rate of 4.2 cm day⁻¹ until the shoot apex reached the pond surface in late April. Both the increment of node number and the internodal elongation were associated with this rapid shoot growth. On 10 May (last sampling date), the mean values of shoot length, internodal length and the number of nodes estimated for 10 predominant plants were 238.2±5.6 cm, 7.1±0.8 cm and 34.9±4.0 cm, respectively. The turion formation and flowering occurred during the period from mid-April to mid-May when the surface water temperature ranged 19 and 22 C. The dry weight of a plant reached the maximum mean value of 1180 mg on 10 May. At its peak biomass, an individual plant produced 1–10 turions (5.5 on average) of which the mean individual turion dry weight was 53.2 mg. The turion dry weight accounted for ca. 42% of the total plant biomass m⁻² at that time.