Assessment, validation and deployment strategy of a two-barcode protocol for facile genotyping of duckweed species.

Lemnaceae, commonly called duckweeds, comprise a diverse group of floating aquatic plants that have previously been classified into 37 species based on morphological and physiological criteria. In addition to their unique evolutionary position among angiosperms and their applications in biomonitoring, the potential of duckweeds as a novel sustainable crop for fuel and feed has recently increased interest in the study of their biodiversity and systematics. However, due to their small size and abbreviated structure, accurate typing of duckweeds based on morphology can be challenging. In the past decade, attempts to employ molecular barcoding techniques for species assignment have produced promising results; however, they have yet to be codified into a simple and quantitative protocol. A study that compiles and compares the barcode sequences within all known species of this family would help to establish the fidelity and limits of this DNA-based approach. In this work, we compared the level of conservation between over 100 strains of duckweed for two intergenic barcode sequences derived from the plastid genome. By using over 300 sequences publicly available in the NCBI database, we determined the utility of each of these two barcodes for duckweed species identification. Through sequencing of these barcodes from additional accessions, 30 of the 37 known species of duckweed could be identified with varying levels of confidence using this approach. From our analyses using this reference dataset, we also confirmed two instances where mis-assignment of species has likely occurred. Potential strategies for further improving the scope of this technology are discussed.

Specific turion yields of different clones of Spirodela polyrhiza depend on external phosphate thresholds.

Turions play an important role in the survival strategy of the duckweed Spirodela polyrhiza. Therefore, factors influencing the formation of these survival organs were studied. Phosphate deficiency is the main natural factor inducing turion formation and the specific turion yield (SY), i.e. the number of turions formed per frond, varied widely for five different clones from different climate zones. The concentrations of phosphate and nitrate in the nutrient media were investigated at the onset of turion formation, with SY ranging from 0.22 to 5.9. Tissue P and N content was also investigated in vegetative fronds at the onset of turion formation and in newly formed turions. The clones were selected to test possible correlations between SY and threshold nutrient concentration for turion formation. Only one correlation, between SY and external phosphate concentration, was significant: clones with high SY started turion formation at higher external phosphate concentrations. Turion formation is thus mainly induced by the external phosphate concentration, below a defined, clone-dependent threshold. We propose the following mechanism: a switch of the developmental programme of frond primordia from vegetative frond to turion formation at a higher phosphate threshold saves more phosphate for turion formation instead of using it for a further vegetative growth. However, the period of growth preceding turion formation does not depend on this threshold value, but rather on the growth rate of the vegetative fronds, which actually produce the phosphate shortage by taking it up from the surrounding medium.

Relative in vitro growth rates of duckweeds (Lemnaceae) - the most rapidly growing higher plants.

Relative growth rates (RGR), doubling times (DT) and relative weekly yields (RY) of 39 clones (ecotypes) from 13 species representing all five genera of duckweeds were determined under standardised cultivation conditions. RGR ranged overall from 0.153 to 0.519 day(-1) , DT from 1.34 to 4.54 days and RY from 2.9 to 37.8 week(-1) . The RGR and RY data can be compared directly to other published findings to only a limited extent on account of missing clonal designations for and limited accessibility to previously investigated clones, as well as the use of different data denominators. However, they are consistent with the published results of other comparative duckweed studies of similar scope in showing that RGR does not vary primarily at the level of the genus or species, but rather reflects the adaptation of individual clones to specific local conditions. The RGR data support the widely held assumption that duckweeds can grow faster than other higher plants and that they can thus surpass land-based agricultural crops in productivity. Duckweeds are highly promising for the production of biomass for nutrition and energy, but extensive clonal comparison will be required to identify the most suitable isolates for this purpose.

The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle.

The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.

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.

A novel mechanism of abscission in fronds of Lemna minor L. and the effect of silver ions.

Lemna minor L. (duckweed) forms colonies through vegetative propagation because mother fronds remain connected for some time with their daughter fronds by stipes. The colony size is controlled by abscission of stipes at a specific preformed abscission zone. Application of silver ions (Ag(+) ) enhances the rate of frond abscission, thus resulting in smaller colonies. The mechanism behind this process has not yet been identified. Silver caused an abscission response that saturated after 7 h of treatment. The half-maximal effective concentration was 0.72 µm Ag(+) for the standard clone, L. minor St. Other clones of the same species show sensitivities that differ by one order of magnitude. Transmission electron microscopy revealed: (i) large numbers of vesicles close to the plasmalemma in cells adjacent to the abscission zone, which proves a vesicular type secretory activity; and (ii) a moderately electron-dense secretion accumulated in the enlarging intercellular spaces, and seemed to flow from the adjacent cells towards the abscission zone. We assume that increasing pressure causes this material to push apart the cells, thereby causing the break in the abscission zone of the stipe. This is a novel mechanism of abscission that has not previously been described. The same mechanism occurs in stipes of both control and Ag(+) -treated samples. Silver ions only accelerate the process leading to abscission of stipes, without affecting the mechanism involved.

Effects of nickel on the chloroplasts of the duckweeds Spirodela polyrhiza and Lemna minor and their possible use in biomonitoring and phytoremediation.

Toxic effects of Ni(2+) on the chloroplasts of the two duckweed species Spirodela polyrhiza, clone SJ and Lemna minor, clone St were investigated according to the ISO 20079 protocol. Ni(2+) induced a transition from chloroplasts to chloro-amyloplasts and amylo-chloroplasts, but not to gerontoplasts, as shown by electron microscopy. The contents of the chlorophylls a and b decreased strongly, whereas that of carotenoids remained approximately constant. Most striking was, however, the accumulation of transitory starch. Bell-shaped dose-response curves showed that Spirodela and Lemna amassed maximum starch contents of approximately 10% and 7%, respectively, on a fresh weight basis. Because Ni(2+) in the concentrations applied does not stimulate photosynthesis, the Ni(2+)-induced starch accumulation indicates that the export of carbohydrates out of the plastids decreased, most probably due to the lower demand of the rest of the cells as a result of the Ni(2+)-dependent inhibition of growth. The half-maximal concentrations for inhibition of the fresh weight increase over the 7-day test period were 3.7 microM and 6.6 microM for Spirodela and Lemna, respectively: Spirodela was thus somewhat more sensitive to the heavy metal. Both species accumulated approximately 3g of Ni(2+) per kg of dry weight after application of 100 microM NiCl(2). Because of their high sensitivity to phytotoxic effects, however, Spirodela and Lemna do not appear to be particularly suitable for phytoremediation of Ni(2+)-contaminated waste water. The high sensitivity to Ni(2+) makes them instead a suitable system for ecotoxicological testing in accordance with the ISO 20079 protocol.

Physiological responses of Juncus effusus (Rush) to chromium and relevance for wastewater treatment in constructed wetlands.

Constructed wetlands are increasingly applied for industrial wastewater treatment. However, current knowledge of the stress responses of helophytes to selected toxicants such as dichromate is limited. The goal of the experiments presented here was to characterize the physiological response of Juncus effusus to different concentrations of dichromate dependent upon the growth and constitution of the plants. The growth parameters, shoot length, and dry weight already were strongly affected at low dichromate concentrations of approximately 34 microM. Concentrations of 340 microM impaired chlorophyll fluorescence and a decrease in chlorophyll a started at concentrations higher than 170 microM dichromate. The concentrations of chlorophyll b and carotenoids also were influenced negatively. Thus, the reduction of the pigment content started before any obvious influence on the chlorophyll fluorescence. The highest concentration of dichromate, which caused no permanent inhibition of growth and photosynthesis, was found to be 17 microM K2Cr2O7. In principle, J. effusus is suitable for constructed wetlands to treat chromium-containing wastewater. Because the stress resistance of J. effusus is limited, the maximum concentration of dichromate in the treated wastewater should not exceed 34 microM. The growth parameters, shoot length, and dry weight were sensitive to much lower dichromate concentrations and did react more quickly than the biochemistry-related parameters chlorophyll fluorescence and pigment concentration. Therefore, the example of Juncus effusus shows that the use of only biochemical parameters to define concentration limits for the treatment of dichromate-containing wastewater can lead to incorrect conclusions and result in disturbed long-term operation of the system.

[Changes in vectors of endogenously generated ion currents in light-induced germination of turions of Spirodela polyrrhiza]. / Izmenenie vektorov éndogenno-generiruemykh ionnykh tokov pri svetoindutsirovannom prorastanii turionov 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.

Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements.

Chromate (Cr) decreases the growth of Spirodela polyrhiza. The fronds lost their pigments. The O2 evolution was also decreased. The Cr effect was found to be dose dependent. The toxic effects of Cr have further been studied on the photosynthetic activity of Spirodela polyrhiza by means of the chlorophyll a (Chl a) fluorescence transient O-J-I-P. The Chl a fluorescence transients were recorded in vivo with high time resolution and analyzed according to the JIP-test which can quantify the photosystem II behavior. Cr treated plants show a decrease in yield for primary photochemistry, phi Po. The performance index of PSII, PIABS, which is the combination of the indexes of three independent parameters, (1) the total number of active reaction centers per absorption (RC/ABS), (2) yield of primary photochemistry (phi Po) and (3) efficiency with which a trapped exciton can move an electron into the electron transport chain (psi 0), decreased due to Cr treatment. Chromate sensitivity varies within plant populations. In summary Cr affects several targets of PSII. More specifically, the main targets of Cr, according to the JIP-test, can be listed as a decrease in the number of active reaction centers and damage to the oxygen-evolving complex.

Light-induced starch degradation in non-dormant turions of Spirodela polyrhiza.

Continuous red light controls starch degradation in turions of Spirodela polyrhiza [Dölger, K., U. K. Tirlapur and K.-J. Appenroth [1997] Photochem. Photobiol. 66, 126-127 (1997)]. This light could be replaced by repeated red light pulses with the reciprocity law fulfilled over a large range of fluence rates. The effect of red light pulses repeated every 24 or 12 h for 6 days was reversible by subsequent far-red light pulses. In contrast, hourly applied red pulses were irreversible by far-red light. This discrepancy was explained by showing the starch degradation activity of far-red pulses themselves. The investigated process was categorized as a phytochrome low fluence response with an unusual property: requirement of light treatment for several days. A partial fulfillment of this requirement was obtained with a red pulse followed by a dark period and a 24 h continuous irradiation. These results suggest the existence of two separate steps in the process of starch degradation in turions: formation of a sprout (= sink) during the pulse-induced germination, and starch degradation in the storage tissue (= source) induced by the second light treatment.

Kinetics of chromium(V) formation and reduction in fronds of the duckweed Spirodela polyrhiza--a low frequency EPR study.

The uptake of chromate by the duckweed Spirodela polyrhiza was investigated with atomic absorption spectroscopy and the reduction of Cr(VI) to Cr(V) was measured using low frequency EPR spectroscopy. The biphasic kinetics of the uptake was fitted to parameters of a proposed kinetic model. Another model was developed to simulate chromate reduction. The first step of chromate reduction was found to be much faster than the uptake of Cr(VI) from the free space. Most probably, this step occurs already in the cell wall or on the cell membrane surface. Further reduction of Cr(V) to Cr(III) was estimated to be slower. The disappearance of the Cr(V) signal, following transfer of the plants into a Cr-free solution, lasted several tens of hours; the kinetics was mono- or biexponential depending on the length of Cr loading. The rate constants for Cr reduction in living plants were determined for the first time.

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 ß-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 ß-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 ß-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 ß-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.

Is there a third photoreceptor involved in the control of chloroplast movements in mougeotia?

The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor.