Turions Of Spirodela Polyrhiza Research Articles

Light-induced Starch Degradation in Non-dormant Turions of Spirodela polyrhiza¶

Light-induced Starch Degradation in Non-dormant Turions of Spirodela polyrhiza¶

Are NADP-dependent isocitrate dehydrogenases and ferredoxin-dependent glutamate synthase co-regulated by the same photoreceptors?

Activities of NADP-dependent isocitrate dehydrogenases (cytosolic and plastidic isoforms, ICDH1 and ICDH2; EC 1.1.1.42) and ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in turions of Spirodela polyrhiza were all stimulated by light. Single or repeated red light (R) pulses induced the activity of the enzymes and this effect was reverted by subsequent far-red light (FR) pulses. The enzymes are, therefore, co-regulated by the low-fluence response of phytochrome. For ICDH, this is reported here for the first time. Neither an effect of the very low-fluence response nor of the FR-mediated high-irradiance response was detectable. Irradiance with continuous R resulted in enhanced enzyme activities and protein levels (Western analysis using polyclonal antibodies against ICDH1 and Fd-GOGAT). These additional effects of continuous R (called a "non-induction effect") could be inhibited for ICDH1 and ICDH2 by the inhibitor of photosynthetic electron transport, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, and are therefore related to the effect of photosynthesis. In contrast, the non-induction effect of Fd-GOGAT was resistant against this inhibitor. Moreover, hourly R pulses did not replace the effect of continuous R. The non-induction effect of light on the activity and protein level of Fd-GOGAT was therefore tentatively classified as an R-mediated high-irradiance response. The activity of Fd-GOGAT but not that of ICDHs was additionally regulated by a specific blue-light receptor. It can be concluded that the levels of ICDHs and Fd-GOGAT were coordinated by light but were not co-regulated by the same photoreceptors. Nitrate is necessary for the light regulation of both enzymes, contributing to the coordinated expression of the relevant genes.

Ion antagonism between calcium and magnesium in phytochrome-mediated degradation of storage starch in Spirodela polyrhiza

Light and calcium regulation were studied in two processes running parallel in turions of Spirodela polyrhiza (L.) Schleiden: in germination and starch degradation. When germination of turions is induced by a single red light (R) pulse the response depends on exogenously applied Ca 2+. This Ca 2+ dependence is absent in continuous and intermittent R (hourly applied short R pulses that can substitute for continuous R). Phytochrome-mediated degradation of storage starch in the turions starts after the onset of germination. The degradation proceeds only in continuous or intermittent R. Contrary to germination, a single R pulse is ineffective in inducing starch degradation. The process, categorized as a special low-fluence phytochrome response, requires Ca 2+. Moreover, a Ca 2+/Mg 2+ ion antagonism was detected: significant starch degradation proceeds in the absence of both Ca 2+ and Mg 2+. Inhibitors of Ca 2+-uptake (La 3+, verapamil) and of calmodulin (chlorpromazine, trifluoperazine) diminish the light-induced starch degradation. This suggests that the antagonistic activity of Ca 2+ and Mg 2+ in this process is associated with the signal transduction chain, similar to that in the R pulse-induced turion germination. Because the Ca 2+ requirement is different in germination and starch degradation, the Ca 2+-sensitive step cannot be the same in the two responses. This Ca 2+/Mg 2+ antagonism can be used to clarify the relationship between biochemical reactions of the composed response of starch degradation and the mode of their regulation.

Association of alpha-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza.

In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein-starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as alpha-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473-477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, alpha-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated alpha-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both alpha-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in vitro studies in which the binding of purified beta-amylase (EC 3.2.1.2) to starch granules isolated from turions following various pretreatments was monitored. The enzyme did bind to starch granules prepared from dark-stored turions (in which starch degradation had not been initiated), but not to those isolated from illuminated (starch degrading) turions.

Light-induced Germination and Endogenous Ion Currents in Turions of Spirodela polyrhiza

Summary Non-dormant turions of the duckweed Spirodela polyrhiza (L.) Schleiden were used to investigate endogenous ion currents in light-induced germination and early growth of higher plants. Before light treatment, a small outward current was measured 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. Dormant turions showed no significant light-induced endogenous current. After germination, young sprouts of Spirodela showed highest inward current near the sprout base where cells grew rapidly and divide frequently. Ageing of sprouts resulted in a fast decrease of endogenous current.

Influence of UVB irradiation on nitrate and ammonium assimilating enzymes in Spirodela polyrhiza

The influence of UVB radiation on nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) was investigated in fronds and turions of Spirodela polyrhiza. When white light (4.3 W m −2) was supplemented with UVB (0.55 W m −2; λ max = 311 nm), a 40% transient decrease in the growth rate was observed after 12 h UVB irradiation. In addition, the activities of all enzymes investigated, except for the cytoplasmic isoform of GS, were substantially inhibited (NR, 80%; NiR, 32%; GS, 30%; NADH-GOGAT, 36%; Fd-GOGAT, 54% in green fronds). The extent of the inhibition in fronds as well as in turions is in accordance with the assumption that the light-induced increases of enzyme activities are prevented by simultaneous application of UVB. The uptake of nitrate was not influenced by white light or UVB. As shown by western analysis, a correlation between the enzyme activities and their protein amounts was found. Northern analysis revealed that UVB influenced the transcript accumulation of NR but not of Fd-GOGAT. No obvious correlation was observed between the mRNA and the protein amount. This suggests that UVB damage occurs at the transcriptional and post-transcriptional levels as well as at the protein level.

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.

Different Regulation of β-amylase and Starch Phosphorylase by Light in Dormant and Non-Dormant Turions of Spirodela polyrhiza

Summary The influence of single red light pulses and of continuous red light on the activity of two enzymes, i.e. β-amylase (EC 3.2.1.2) and starch phosphorylase (EC 2.4.1.1), was investigated in dormant and non-dormant turions of Spirodela polyrhiza. The activity of β-amylase was induced by continuous red light but not by red light pulses in dormant turions. In non-dormant turions, however, the activity of β-amylase following a single red light pulse was two times higher than after continuous red light. This high effectiveness of a single red light pulse, as compared with continuous red, represents a novel mode of regulation of enzyme activity by light. It is suggested that a low fluence response of phytochrome is abolished by a high irradiance response elicited by continuous red light. The light-induced increase in the activity of β-amylase is due to de novo synthesis of the enzyme as shown by Western blot analysis using polyclonal antibodies raised against β-amylase from Spirodela polyrhiza. The presence of light-activable precursor was excluded by showing that cysteine added to the extraction medium did not influence the activity of β-amylase. In contrast, the activity of starch phosphorylase was induced by continuous red light in dormant turions but decreased in non-dormant turions. During the cold after-ripening treatment turions qualitatively changed their response to light, becoming light-insensitive when partly after-ripened (21 days). In all instances, a red light pulse was ineffective in inducing starch phosphorylase. Isoforms of β-amylase and starch phosphorylase were separated by non-denaturating PAGE. No special induction by light of any specific isoform of β-amylase and starch phosphorylase was found in turions. Since both enzymatic activities and their regulation by light do not correlate with the breakdown of starch, we conclude that they are not directly involved in the regulation of starch degradation in turions.

Regulation of transcript level and nitrite reductase activity by phytochrome and nitrate in turions of Spirodela polyrhiza

Regulation of transcript level and nitrite reductase activity by phytochrome and nitrate in turions of Spirodela polyrhiza

Regulation of transcript level and nitrite reductase activity by phytochrome and nitrate in turions of Spirodela polyrhiza

Control by light and nitrate of the appearance of nitrite reductase (NIR; EC 1.7.7.1) activity was investigated in turions of Spirodela polyrhiza during the pre‐germination period to establish the nature of the coaction between these factors. A cDNA clone coding for tobacco NIR was available as a probe. In the period immediately after transfer from after‐ripening conditions (5°C. darkness) to germination conditions (25°C; phase 1, from 0 to 72 h after start of the experiments) a nitrate‐dependent increase in NIR activity was observed, presumably because of the temperature shift. During this phase light had no effect on the nitrate‐dependent increase in NIR activity. In contrast, after a pre‐treatment of 72 h in darkness at 25°C following cold afterripening responsiveness to phytochrome appeared, with light inducing a nitrate‐dependent increase in NIR activity (phase II, from 72 to 120 h after start of the experiments). Application of nitrate (10 mM) in phase II also resulted in an increase of NIR activity in light or darkness although highest NIR activities were observed in the presence of light. Ammonium did not affect the appearance of NIR activity. In all experiments, a single isoform of NIR was detectable in both phases. It was concluded that the mode of coaction between light (via phytochrome) and nitrate is different in phase I and phase II and depends on the developmental state of the plant organs. Furthermore, the response patterns of nitrate reductase and NIR activities to both factors is so similar in turions that co‐regulation of both enzymes can be assumed, as found previously in tobacco seedlings. Northern blot analyses revealed a low NIR transcript level in the absence of nitrate in darkness during phase II. Both light and nitrate alone were sufficient to stimulate the NIR mRNA steady state level and the highest transcript level was detectable in the presence of both stimulators. This demonstrates a synergistic effect of both factors on steady state NIR transcript level.

The appearance of glutamine synthetase in turions ofSpirodela polyrhiza(L.) Schleiden as regulated by blueand red light, nitrate and ammonium

Control by light and nitrogen (nitrate and ammonium) of the appearance of glutamine synthetase (GS; EC 6.3.1.2) in turions of Spirodela polyrhiza (L.) Schleiden, strain SJ, was investigated during the pregermination period, i.e. up to 48 h after onset of light. Immediately after transfer from after-ripening conditions (5 o C, darkness, D) to germination conditions (25 o C), GS activity did not respond to light or nitrate. After 72 h in D (25 o C) activity increased in continuous light. Therefore, the regulatory role of light, nitrate and ammonium in the process of appearance of GS was mainly studied between 72 and 120 h after transfer from after-ripening to germination conditions (phase II of the pre-germination process)

Effect of UVB irradiation on enzymes of nitrogen metabolism in turions of Spirodela polyrhiza (L.) Schleiden

The influence of UVB irradiation on the enzymes of nitrogen metabolism (nitrate reductase, EC 1.6.6.1, nitrite reductase, EC 1.7.7.1, alanine aminotransferase, EC 2.6.1.2, aspartate aminotransferase, EC 2.6.1.1, glutamine synthetase, EC 6.3.1.2) and the uptake of ammonium and nitrate in turions of Spirodela polyrhiza was investigated. UVB irradiation was started either immediately after transferring the turions from 5 °C (after-ripening conditions) to 25 °C (germination conditions) or after a storage period at 25 °C in darkness for 72 h. The activities of all enzymes investigated were decreased by UVB irradiation (12 or 24 h). The activity of nitrate reductase was more strongly inhibited than that of nitrite reductase, and the activity of alanine aminotransferase was more strongly decreased than that of aspartate aminotransferase. The inhibitory effect of UVB on glutamine synthetase was only small. Within a period of 5 h neither the uptake of ammonium nor that of nitrate was influenced by UVB irradiation. Because UVB irradiation did not influence the light-dependent germination of turions, the results presented support the conclusion that the regulation of germination and the regulation of nitrate assimilation are unrelated phenomena in turions

Regulation of transcript level and synthesis of nitrate reductase by phytochrome and nitrate in turions of Spirodela polyrhiza (L.) Schleiden

Control by light and nitrate of the appearance of nitrate reductase (NR; EC 1.6.6.1) in the turions of Spirodela polyrhiza (L.) Schieiden was investigated during the pre-germination phase, i.e. up to 120 h after the transfer from after-ripening to germination conditions. Turions are particularily suited for this type of coaction study since control of nitrate uptake by light and long-distance transport of nitrate do not play a role. Control of NR gene expression was mainly studied between 72 and 120 h after transfer to germination conditions (phase II of the pre-germination process). It was found that the effect of light on NR appearance is exerted via phytochrome. The light effect on enzyme synthesis was only observed in the presence of nitrate. On the other hand, in darkness, the NR level decreased to almost zero even in the presence of an optimum supply of nitrate. It is concluded that in phase II a coaction of light (phytochrome) and nitrate is required to bring about NR synthesis. However, when nitrate was applied to turions - following a dark incubation without nitrate - the turions responded to nitrate even in darkness with strong NR synthesis. This response was augmented by light. The highest transcript levels were observed in the presence of both factors, light and nitrate. As a single factor, light was more effective in stimulating the transcript level than nitrate. However, no correlation between the transcript level and the rate of enzyme synthesis was observed. This is consistent with the previous conclusion (Schuster et al. 1988, Planta 174, 426-432) that in higher plants control at the transcript level is only coarse and does not determine quantitatively the output at the level of enzyme protein.

Photophysiology of turion germination in Spirodela polyrhiza (L.) Schleiden VIII. Calcium dependence and ?-amylase activity

Red-light-induced germination (via phytochrome) of turions of Spirodela polyrhiza (L.) Schleiden, strain SJ, showed an absolute requirement for exogenously applied calcium (Appenroth et al. 1990b, Biochem. Physiol. Pflanzen 186, 209-219). With calcium step-up experiments (from 0.9 μM to 1 mM Ca(2+)) the influence of Ca(2+) on the escape from far-red-light reversibility was investigated. Delaying the time of Ca(2+) application after the red light pulse resulted in an increased reversibility. This confirmed that the calcium-requiring phase is within the phytochrome-dependent period. Ten days after a red light pulse was applied the activity of β-amylase (EC 3.2.1.2) was increased about tenfold. The starting point was similar in the germination and enzymatic responses, but half-maximal response times according to the Mitscherlich function (2.5 d and 6.9 d, respectively) and times of saturation (about 6 d and 9 d, respectively) were different. Increase of β-amylase activity was completely inhibited by irradiation with far-red light (demonstrating phytochrome as the photoreceptor), as well as by inhibitors of translation (cycloheximide, puromycin) and transcription (cordycepin). Once formed, the active enzyme protein was stable for at least 4 d after cycloheximide application. After application of cordycepin a small amount of translation activity was detected until complete inhibition was reached after 2 d. This phytochrome-modulated, de-novo synthesis of β-amylase was only slightly dependent on exogenous Ca(2+). Therefore, it can be concluded that the phytochrome-mediated signal-transduction chain branches either in its reversible part or as a consequence of diversity of the primary phytochrome reaction.

Photophysiology of Turion Germination in Spirodela polyrhiza (L. ) SCHLEIDEN. Iv. Importance of Calcium and Calmodulin

Light-grown as well as dark-grown (etiolated) turions of Spirodela polyrhiza are positively photoblastic. The phytochrome-mediated germination response requires supply with exogenous calcium (Ca 2+ ). Threshold value is 1.3 ± 0.3 µM Ca 2+ , half-maximal response is mediated by 16 ±2 µM Ca 2+ . Ca 2+ may be replaced in part by Sr 2+ , but not by Mg 2+ . Furthermore, the addition of the Ca 2+ -antagonists La 3+ and Co 2+ causes a complete inhibition of germination. Delayed addition of La 3+ up to 72 h after the red light pulse results in a decreased, but still significant inhibition. Germination is also inhibited by the Ca 2+ -channel blocker verapamil. On the other hand, stimulation of germination is observed without red light pulses if the Ca 2+ -ionophore A 23187 is applied for 24 h. Complete inhibition of germination is caused by the calmodulin inhibitors chlorpromazine and trifluoperazine, and, besides, calmodulin was detected in the turions. Consequently, Ca 2+ as well as calmodulin are involved in the phytochrome-mediated germination response of turions.

Influence of Nutrient Deficiency and Light on Turion Formation in Spirodela polyrhizass (L.) SCHLEIDEN

Turions of Spirodela polyrhiza were formed under nitrate-limiting, mixotrophic (N-mixo), sulfatelimiting, mixotrophic (S-mixo) and phosphate-limiting, mixotrophic (P-mixo), heterotrophic (P-hetero) and autotrophic (P-auto) conditions in continuous white light.Yield of turions (harvested 28 days after inoculation; P-auto after 50 days) was found as follows: S-mixo > P-mixo > P-hetero > P-auto > N-mixo. Concerning turion width and uniformity (reciprocal variance of width) the following series were obtained: P-hetero > P-mixo > P-auto, N-mixo > P-mixo > S-mixo, and P-hetero > P-mixo > P-auto, N-mixo ≈ P-mixo > S-mixo, respectively. P-mixo-conditions represent good turion yield and morphological uniformity. Production of large amounts of P-hetero-turions in a short period of time was attained by subsequent heterotrophic cultivation of mixotrophically precultivated fronds. As compared with P-mixo-turions, P-hetero-turions had a higher content of water, total nitrogen, soluble protein and ethanol-soluble carbohydrates. By contrast, in Phetero-turions content of chlorophyll and all carotenoids was found to be lower.