Antagonism or synergism? Responses of Hydrocharis dubia (Bl.) Backer to linear alkylbenzene sulfonate, naphthalene and their joint exposure.

The presence of surfactants may affect the bioavailability of polycyclic aromatic hydrocarbons. A hydroponic experiment was conducted to investigate the response of Hydrocharis dubia (Bl.) Backer to different concentrations of linear alkylbenzene sulfonate (LAS), naphthalene (NAP) and their mixture (0.5, 5, 10, and 20 mg/L) for 14 days and 28 days. The results showed that LAS had a greater toxic effect on H. dubia growth than NAP at treatment concentrations of 0.5-20 mg/L. The combined effect of LAS and NAP was damaging to H. dubia at concentrations of LAS + NAP ≥5 + 5 mg/L. When LAS + NAP ≥10 + 10 mg/L, the underground parts of H. dubia suffered more significant damage than the aboveground parts. Under the treatments with LAS, NAP and their mixture, H. dubia experienced oxidative stress. Soluble proteins and antioxidant enzymes were the main substances protecting H. dubia from LAS stress, and superoxide dismutase (SOD) and peroxidase (POD) were the main protective enzymes. When exposed to NAP, H. dubia growth was stimulated and promoted at the same time. In the short-term treatment (14 d), catalase (CAT) activity was sensitive to NAP stimulation, and soluble proteins and SOD were the main protective substances produced. Soluble sugars, SOD and ascorbate peroxidase (APX) played important protective roles during the longer exposure time (28 d). The physiological response of H. dubia exposed to the combined toxicants was weaker than the response to exposure to individual toxicants. The responses of SOD and CAT activity were positive in the short term (14 d), and these were the main protective enzymes. As the exposure time increased (28 d), the plant antioxidant system responded negatively.

Subcellular accumulation and source of O2- and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle under NH4+-N stress condition.

In this study, the effects of excess NH4+-N on the subcellular accumulation of O2- and H2O2 in submerged plant Hydrilla verticillata (L.f.) Royle were investigated using both histochemical and cytochemical methods. Treatments with ≥ 2.00 and ≥ 5.00 mg L-1 NH4+-N for 5 d significantly increased production of O2- and H2O2, respectively. The activities of plasma membrane-bound NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and antioxidant enzymes (superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, dehydroascorbate reductase and glutathione reductase) were also increased correspondingly. This study also provides the first cytochemical evidence of subcellular accumulation of O2- and H2O2 in the submerged plants. In the leaves of H. verticillata treated with 20.0 mg L-1 NH4+-N, O2- dependent DAB precipitates were found primarily on the inner side of the plasma membrane, extracellular space and chloroplasts. H2O2-CeCl3 precipitates were mainly localized on the inner side of the plasma membrane and extracellular space of the mesophyll cells. Treatments with the inhibitors of NADPH oxidase (diphenylene iodonium and imidazole) indicate that NH4+-N-induced production of O2- and H2O2 in H. verticillata leaves may involve plasma membrane-bound NADPH oxidase. Moreover, low-light treatment decreased NH4+-induced O2- production, suggesting that alterations in the photosynthetic electron transfer chain due to NH4+ toxicity could lead to O2- production.

Different CO2 acclimation strategies in juvenile and mature leaves of Ottelia alismoides.

The freshwater macrophyte, Ottelia alismoides, is a bicarbonate user performing C4 photosynthesis in the light, and crassulacean acid metabolism (CAM) when acclimated to low CO2. The regulation of the three mechanisms by CO2 concentration was studied in juvenile and mature leaves. For mature leaves, the ratios of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) are in the range of that of C4 plants regardless of CO2 concentration (1.5-2.5 at low CO2, 1.8-3.4 at high CO2). In contrast, results for juvenile leaves suggest that C4 is facultative and only present under low CO2. pH-drift experiments showed that both juvenile and mature leaves can use bicarbonate irrespective of CO2 concentration, but mature leaves have a significantly greater carbon-extracting ability than juvenile leaves at low CO2. At high CO2, neither juvenile nor mature leaves perform CAM as indicated by lack of diurnal acid fluctuation. However, CAM was present at low CO2, though the fluctuation of titratable acidity in juvenile leaves (15-17 µequiv g-1 FW) was slightly but significantly lower than in mature leaves (19-25 µequiv g-1 FW), implying that the capacity to perform CAM increases as leaves mature. The increased CAM activity is associated with elevated PEPC activity and large diel changes in starch content. These results show that in O. alismoides, carbon-dioxide concentrating mechanisms are more effective in mature compared to juvenile leaves, and C4 is facultative in juvenile leaves but constitutive in mature leaves.

Phosphate alleviation of glyphosate-induced toxicity in Hydrocharis dubia (Bl.) Backer.

Glyphosate, as a broad-spectrum herbicide, is frequently detected in water, and phosphorus widely enters the water due to the extensive use of phosphorus-containing substances in agriculture, industries and daily life. Thus, aquatic ecosystems are exposed to both glyphosate and phosphorus, which may affect aquatic organisms. In the present research, we studied the physiological responses of the floating aquatic plant species H. dubia to different concentrations of glyphosate (0, 1, 5, 15 mg/L) with different levels of phosphate (0, 50, 100 mg/L) after 14 days (d) of treatment. We explored glyphosate toxicity in H. dubia and investigated whether phosphate addition mitigates glyphosate toxicity in this species, which will provide a theoretical basis for the ecotoxicological study of aquatic plants. The results show that glyphosate significantly reduced the chlorophyll content, leaf number and root length of H. dubia, while it significantly increased the malondialdehyde (MDA), hydrogen peroxide (H2O2), shikimate, proline, and soluble protein content and enzyme activities (superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and polyphenol oxidase (PPO)) in H. dubia. After phosphate supplement, the MDA, H2O2, proline, and soluble protein contents and enzyme activities in the plants treated with glyphosate decreased. These results indicate that the concentration of glyphosate investigated in our study can cause oxidative stress and affect the growth of H. dubia. Phosphate can alleviate glyphosate-induced oxidative stress in H. dubia.

Response of biofilms-leaves of two submerged macrophytes to high ammonium.

Submerged macrophytes can provide attached surface for biofilms (known as periphyton) growth. In the present study, the alterations in biofilms formation, and chemical compositions and physiological responses were investigated on leaves of Vallisneria asiatica and Hydrilla verticillata exposed to 0.1 mg L-1 (control) or with 10 mg L-1 NH4+-N for 13 days. Results from physiological and biochemical indices (content of H2O2, malondialdehyde, total chlorophyll and activity of superoxide dismutase, catalase and peroxidase) showed that high ammonium caused oxidative damage to leaves of two species of plant. Multifractal analysis (based on scanning electron microscope images) showed that for the same plant, the values of width △α (△α = αmax-αmin) of the f(α) and Δf (Δf = f(αmin)-f(αmax)) were smaller on leaves surface of two species of plant treated with 10 mg L-1 NH4+-N for 13 days than their controls, suggesting high ammonium treatments reduced morphological heterogeneity of leaf surface and enhanced area of the colony-like biofilms. X-ray photoelectron spectroscopy analysis showed that C, O, N and P were dominant elements on leaves surface of two species of plant and ammonium application increased the percentage of C but decreased that of O. High ammonium increased C1 (C-C or C-H) percentage but decreased C2 (C-O) and C3 (O-C-O or C=O) percentage on leaves surface of two species of plant, indicating that ammonium stress changed the surface chemical states and thus might reduce the capacity of leaves to adsorb nutrients from water column. Our results provided useful information to understand ammonium induced toxicity to submerged macrophytes.

Responses of Ottelia alismoides, an aquatic plant with three CCMs, to variable CO2 and light.

Ottelia alismoides is a constitutive C4 plant and bicarbonate user, and has facultative crassulacean acid metabolism (CAM) at low CO2. Acclimation to a factorial combination of light and CO2 showed that the ratio of phosphoenolpyruvate carboxylase (PEPC) to ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (>5) is in the range of that of C4 plants. This and short-term response experiments showed that the activity of PEPC and pyruvate phosphate dikinase (PPDK) was high even at the end of the night, consistent with night-time acid accumulation and daytime carbon fixation. The diel acidity change was maximal at high light and low CO2 at 17-25 µequiv g-1 FW. Decarboxylation proceeded at ~2-3 µequiv g-1 FW h-1, starting at the beginning of the photoperiod, but did not occur at high CO2; the rate was greater at high, compared with low light. There was an inverse relationship between starch formation and acidity loss. Acidity changes account for up to 21% of starch production and stimulate early morning photosynthesis, but night-time accumulation of acid traps <6% of respiratory carbon release. Ottelia alismoides is the only known species to operate CAM and C4 in the same tissue, and one of only two known aquatic species to operate CAM and bicarbonate use.

Antioxidant Responses of Vallisneria asiatica to Eutrophic Sediments in Lake Taihu, China.

Three kinds of representative sediments were obtained from a macrophyte-dominated bay (East Lake Taihu) and two algae-dominated regions (Western Lake Taihu and Meiliang Bay). Physiological responses of Vallisneria asiatica to these sediments were compared. Results from 20 days exposures showed no obvious differences in malondialdehyde (MDA) in roots, while the MDA content in leaves of plants exposed to Western Lake Taihu sediment was significantly (p<0.05) higher than those exposed to the other two sediments. In comparison to the other two sediments, plants exposed to Western Lake Taihu sediment showed significantly lower (p<0.05) superoxide dismutase in roots and leaves on the 10th and 40th day. On the 40th day, root catalase (CAT) activities in V. asiatica from Western Lake Taihu and Meiliang Bay sediments were lower than that from East Lake Taihu sediment, while leaf CAT activity in V. asiatica from Western Lake Taihu sediment was higher than that from East Lake Taihu sediment (p<0.05). Western Lake Taihu sediment caused more serious oxidative stress in V. asiatica than East Lake Taihu sediment. Results indicated eutrophic sediment was a contributing factor in the disappearance of V. asiatica in Western Lake Taihu.

Lipopolysaccharides elicit an oxidative burst as a component of the innate immune system in the seagrass Thalassia testudinum.

This study represents the first report characterizing the biological effects of a lipopolysaccharide (LPS) immune modulator on a marine vascular plant. LPS was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS), detected by a redox-sensitive fluorescent probe and luminol-based chemiluminescence, included a diphenyleneiodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900 bp cDNA fragment coding for this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. The triggered release of ROS occurred at 20 min post-elicitation and was dose-dependent, requiring a minimal threshold of 50 µg/mL LPS. Pharmacological dissection of the early events preceding ROS emission indicated that the signal transduction chain of events involved extracellular alkalinization, G-proteins, phospholipase A2, as well as K(+), Ca(2+), and anion channels. Despite exclusively thriving in a marine environment, seagrasses contain ROS-generating machinery and signal transduction components that appear to be evolutionarily conserved with the well-characterized defense response systems found in terrestrial plants.

Influence of exogenous urea on photosynthetic pigments, (14)CO 2 uptake, and urease activity in Elodea densa-environmental implications.

This paper analyzes the effect of exogenous urea in increased concentration gradient (0, 100, 500 and 1,000 mg L(-1)) on photosynthetic pigments (measured spectrophotometrically), uptake of (14)CO2 (using radioisotope), and urease activity (by measuring ammonia with Nessler's reagent) in leaves of Elodea densa Planch. We have observed that low concentration of urea (100 mg L(-1)) stimulates the accumulation of photosynthetic pigments and intensifies photosynthesis in E. densa, whereas high concentration (1,000 mg L(-1)) suppresses these processes. Urease activity increased by approximately 2.7 and 8 fold when exogenous urea concentrations were 100 and 500 mg L(-1), respectively. However, exogenous urea in high concentration (1,000 mg L(-1)) decreased urease activity by 1.5 fold compared to the control. The necessity of mitigating urea and other nitrogen-containing compounds (NH3 from urea) in water bodies has been discussed with emphasis on the potential for phytoremediation of urea using common water weed viz. E. densa.

Toxic effects of Hydrilla verticillata exposed to toluene, ethylbenzene and xylene and safety assessment for protecting aquatic macrophytes.

Little information is available about the toxicity of toluene, ethylbenzene and xylene acting on macrophytes, and their toxicity data are rarely used in regulation and criteria decisions. The results extended the knowledge on toxic effects of toluene, ethylbenzene and xylene on aquatic plants. The responses of Hydrilla verticillata to these pollutants were investigated. Chlorophyll levels, lipid peroxidation, and antioxidant enzymes (superoxide dismutase and guaiacol peroxidase) showed diverse responses at different concentrations of toluene, ethylbenzene and xylene. The linear regression analyses were performed respectively, suggesting the concentrations of toluene, ethylbenzene and xylene expected to protect aquatic macrophytes were 7.30 mg L⁻¹, 1.15 mg L⁻¹ and 2.36 mg L⁻¹, respectively. This study emphasized that aquatic plants are also sensitive to organic pollutants as fishes and zooplanktons, indicating that macrophytes could be helpful in predicting the toxicity of these pollutants and should be considered in regulation and criteria decisions for aquatic environment protection.

Lessons from engineering a single-cell C(4) photosynthetic pathway into rice.

The transfer of C(4) plant traits into C(3) plants has long been a strategy for improving the photosynthetic performance of C(3) plants. The introduction of a pathway mimicking the C(4) photosynthetic pathway into the mesophyll cells of C(3) plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C(4)-like pathway in which CO(2) capture and release occur in the mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and 12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these studies nonetheless have important implications for the evolution of C(4) photosynthetic genes and their metabolic regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes the lessons learned during these attempts to engineer single-cell C(4) rice.

Enzymatic basis for fungicide removal by Elodea canadensis.

PURPOSE: Plants can absorb a diversity of natural and man-made toxic compounds for which they have developed diverse detoxification mechanisms. Plants are able to metabolize and detoxify a wide array of xenobiotics by oxidation, sugar conjugation, glutathione conjugation, and more complex reactions. In this study, detoxification mechanisms of dimethomorph, a fungicide currently found in aquatic media were investigated in Elodea canadensis. METHODS: Cytochrome P450 (P450) activity was measured by an oxygen biosensor system, glucosyltransferases (GTs) by HPLC, glutathione S-transferases (GSTs), and ascorbate peroxidase (APOX) were assayed spectrophotometrically. RESULTS: Incubation of Elodea with dimethomorph induced an increase of the P450 activity. GST activity was not stimulated by dimethomorph suggesting that GST does not participate in dimethomorph detoxification. In plants exposed to dimethomorph, comparable responses were observed for GST and APOX activities showing that the GST was more likely to play a role in response to oxidative stress. Preincubation with dimethomorph induced a high activity of O- and N-GT, it is therefore likely that both enzymes participate in the phase II (conjugation) of dimethomorph detoxification process. CONCLUSIONS: For the first time in aquatic plants, P450 activity was shown to be induced by a fungicide suggesting a role in the metabolization of dimethomorph. Moreover, our finding is the first evidence of dimethomorph and isoproturon activation of cytochrome P450 multienzyme family in an aquatic plant, i.e., Elodea (isoproturon was taken here as a reference molecule). The detoxification of dimetomorph seems to proceed via hydroxylation, and subsequent glucosylation, and might yield soluble as well as cell wall bound residues.

Effect of variable sulfur supply on arsenic tolerance and antioxidant responses in Hydrilla verticillata (L.f.) Royle.

In the present study, Hydrilla verticillata plants were exposed to arsenate (AsV; 50 microM) and arsenite (AsIII; 5 microM) under variable S supply: deficient (2 microM S, -S), normal (1 mM S, +S) and excess (2 mM S, +HS). Arsenic accumulation (microg g(-1) dw) in +HS plants was about 2-fold higher upon exposure to both AsV (30) and AsIII (50) than that observed in +S (12 & 24) and -S (14 & 26) plants. Despite lower As accumulation, -S plants experienced the maximum oxidative stress owing to an inadequate response of enzymatic and molecular antioxidants and significant decline in total thiols and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). By contrast +HS plants had significant increase in total thiols and an improved redox status, did not demonstrate any negative impact to antioxidants except catalase and hence experienced the least increase in oxidative stress parameters. In conclusion, an increase in S supply to plants may improve their accumulation capacity for As through enhanced tolerance caused by a positive effect on thiol metabolism and antioxidant status of the plants.

Effects of ammonium on the antioxidative response in Hydrilla verticillata (L.f.) Royle plants.

To investigate ammonium toxicity, the submerged plant Hydrilla verticillata (L.f.) Royle was treated with 0.1-3.0mM ammonium for 12h and 4d. After exposure to ammonium for 4d, content of O2(-) and H2O2 increased in leaves of H. verticillata exposed to 3mM ammonium compared with control (0mM NH4Cl), while the malondialdehyde content decreased. The chlorophyll (a+b) and carotenoid concentrations decreased in H. verticillata plants exposed to 1.5-3mM ammonium for 12h and 4d. Compared with controls, the activity of superoxide dismutase, peroxidase, catalase, glutathione reductase, ascorbate peroxidase, and glutathione S-transferase increased in plants treated with ammonium for 12h, and the activity of most enzymes was further enhanced at 4d. The changes in nonprotein thiols, total glutathione, ascorbic acid, and dehydroascorbate content were also assayed. Our results suggest that ammonium induced the oxidative stress and the heated antioxidant response in H. verticillata.

Increasing sulfur supply enhances tolerance to arsenic and its accumulation in Hydrilla verticillata (Lf.) Royle.

The present study was aimed to analyze the effects of variable S supply on arsenic (As) accumulation potential of Hydrilla verticillata (Lf.) Royle. Plants were exposed to either arsenate (AsV; 50 microM) or arsenite (AsIII; 5 microM) for 4 h and 1 day while S supply was varied as deficient (2 microM, -S), normal (1 mM, +S) and excess (2 mM, +HS). The level of As accumulation (microg g(-1) dw) after 1 day was about 2-fold higher upon exposure to either AsV (30) or AsIII (50) in +HS plants than that being in +S (12 and 24) and -S (14 and 26) plants. The +HS plants showed a significant stimulation of the thiol metabolism upon As exposure. Besides, they did not experience significant toxicity, measured in terms of malondialdehyde accumulation; an indicator of oxidative stress. By contrast, -S plants suffered from oxidative stress probably due to negative impact to thiol metabolism. Variable S supply also modulated the activity of enzymes of glycine and serine biosynthesis indicating an interconnection between S and N metabolism. In conclusion, an improved supply of S to plants was found to augment their ability for As accumulation through stimulated thiol metabolism.

Characterization of the NADP malic enzyme gene family in the facultative, single-cell C4 monocot Hydrilla verticillata.

Hydrilla verticillata has a facultative single-cell system that changes from C3 to C4 photosynthesis. A NADP+-dependent malic enzyme (NADP-ME) provides a high [CO2] for Rubisco fixation in the C4 leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C4 leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C4 species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C3 and C4 species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The Km (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C4 system in H. verticillata.

[Protective effects of exogenous ascorbic acid on antioxidant system in Hydrilla verticillata under Cd2+].

In this paper, Hydrilla verticillata was cultured in the hoagland solution containing 5 mg L(-1) Cd2+ and different concentration ascorbic acid (AsA) to investigate the protective action of AsA to aquatic plant suffered from heavy metal's stress. The O2 generation rate, H2O2 content, antioxidants (AsA and GSH) contents, and antioxidant enzymes (SOD, POD, CAT and APX) activities were analyzed. The results showed that compared with single Cd2+ treatment, AsA addition lessened the reactive oxygen species (O2-* and H2O2) generation rates, endogenous AsA content, and antioxidant enzyme activities. It was concluded that exogenous AsA could relieve the Cd2+ poison to H. verticillata, and its optimum concentration was 60 mg x L(-1).

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker.

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.

Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle.

Copper, though essential, is potentially toxic heavy metal at supraoptimal level and has widespread contamination. The present investigation was carried out to study the responses induced by lower as well as higher doses of copper (0.1-25 microM) in an aquatic macrophyte, Hydrilla verticillata (L.f.) Royle for a period of 1-7 days. The plants accumulated copper in high amount with a maximum of 770 microg g(-1) dw on day 7 at 25 microM. Biomass and photosynthetic pigments showed less alteration up to 1 microM while at higher concentrations, significant decline occurred. Malondialdehyde (MDA) content and electrical conductivity (EC) also showed sharp increase at higher concentrations indicating oxidative stress. In response to copper exposure, plants showed significant induction of proteins and enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR), however, only up to moderate exposures. Total non-protein thiols (NP-SH) and cysteine levels increased significantly up to 5 microM copper exposure while at 25 microM, their level declined drastically. Reduced glutathione (GSH) showed decrease at all concentrations while oxidized glutathione (GSSG) simultaneously increased. Phytochelatins (PCs) were also induced significantly at studied concentrations of 1 and 5 microM on day 4 in comparison to control. However, copper chelation depicted by PC-SH to copper ratio was found to be low (6.5% at 1 microM and 2.4% at 5 microM) suggesting that PCs play only a part in integrated mechanisms of copper homeostasis and detoxification. Tolerant response of plants to moderate copper exposures and high accumulation potential warrants their suitability for remediation of moderately copper polluted water bodies.

Light regulation of the photosynthetic phosphoenolpyruvate carboxylase (PEPC) in Hydrilla verticillata.

The submersed monocot, Hydrilla verticillata (L.f.) Royle, is a facultative C(4) NADP-malic enzyme (NADP-ME) plant in which the C(4) and Calvin cycles co-exist in the same cell. Futile cycling is avoided by an intracellular separation of carboxylases between the cytosol and chloroplasts. Of the two sequenced H. verticillata phosphoenolpyruvate carboxylase (PEPC) isoforms, hvpepc3 and hvpepc4, transcript expression of the latter was substantially up-regulated during C(4) induction, especially in the light. Western blots revealed two PEPC-specific bands in C(3) and C(4) leaf extracts; the lower band dominated in the C(4) and underwent post-translational phosphorylation in the light as determined by immunological studies. This band probably represents the photosynthetic isoform, HVPEPC4, despite the lack of the C(4) signature serine (Flaveria residue 774; Hydrilla 779). In C(4) leaves, PEPC activity increased 14-fold, was enhanced by leaf exposure to light, and showed allosteric regulation. Glucose-6-phosphate acted as a positive effector, but malate was inhibitory, with I(50) values of 0.4 and 0.2 mM in the light and dark, respectively, similar to those of other C(4) PEPC isoforms. In contrast, in C(3) leaves, transcript expression of both isoforms was weak, with little evidence of diel regulation, and the PEPC proteins showed essentially no indication of phosphorylation. PEPC activity in C(3) leaves was low, light independent and followed Michaelis-Menten kinetics. It was tolerant to malate, with 10-fold higher I(50) values than the PEPC from C(4) leaves. These data suggest that hvpepc4 encodes the C(4) photosynthetic PEPC, and hvpepc3 encodes an anaplerotic form.