PSII Inhibitors Research Articles

Application of fast chlorophyll a fluorescence kinetics to probe action target of 3-acetyl-5-isopropyltetramic acid

3-Acetyl-5-isopropyltetramic acid (3-AIPTA), an analogue of the phytotoxin tenuazonic acid, is a tetramic acid derivate. It is demonstrated here that 3-AIPTA is a multi-target inhibitor of root and shoot growth as well as photosynthesis. Based on fast chlorophyll fluorescence kinetics, 3-AIPTA blocks electron transport beyond QA on the acceptor side of PSII by competing with QB for the QB-binding site, but does not affect the donor side and the light harvesting function of the PSII antenna. Additionally, higher 3-AIPTA concentration also inhibits the reduction of end acceptors of PSI. Evidences from JIP-test and competitive replacement with [14C]atrazine showed that 3-AIPTA, like tenuazonic acid, does not share the same binding environment with the atrazine-like PSII inhibitors although they possess the common action target of the QB-site. It is deduced that tetramic acid families of natural products, where 3-AIPTA and tenuazonic acid belong to, is a novel inhibitor type of the photosynthetic electron transport chain.

Effect of prolonged hypoxia in autotrophic conditions in the hydrogen production by the green microalga Chlamydomonas reinhardtii in photobioreactor

In the context of hydrogen production by the green alga Chlamydomonas reinhardtii, the control of light attenuation conditions is used to set-up anoxia under illuminated and autotrophic conditions, without affecting photosynthetic capacities of cells (as with sulphur deprivation or PSII inhibitors like DCMU). This paper presents a full description of the protocol where the incident photons flux density (PFD) is adapted during cultivation in order to obtain a sufficiently low illuminated fraction γ under 0.25 leading to anoxic hydrogen producing conditions during several days. The protocol is validated in a torus-shape photobioreactor (PBR) revealing after few days of anoxic conditions a peak of hydrogen production (1.44 ml H2/h/l of culture; [0.8-1.0] ml H2/h/g of dry weight biomass) concomitant with a decrease of biomass concentration, protein content and maximal photosynthetic yield. Effect of over-accumulating starch, as being known to increase hydrogen production by the PSII-independent pathway, is also investigated.

Molecular Analysis of Hexazinone-Resistant Shepherd's-Purse (Capsella bursa-pastoris) Reveals a NovelpsbAMutation

A population of shepherd's-purse suspected to be resistant to the triazinone herbicide hexazinone, a photosystem II (PS II) inhibitor, was collected from an alfalfa field in 2007 in Oregon. A whole-plant, dose–response assay confirmed that the putative-resistant population was highly resistant to hexazinone. The resistant population was 22-fold more resistant to hexazinone than the susceptible population. However, the hexazinone-resistant population was susceptible to other PS II-inhibiting herbicides, including atrazine, diuron, and terbacil. DNA sequence analysis of the chloroplastpsbAgene encoding the D1 protein of PS II, the target site of PS II inhibitors, identified a point mutation from Phe to Ile at position 255 in the hexazinone-resistant population. Single- and double-point mutations at position 255, which is located in the QBbinding niche of the D1 protein, were previously reported inChlamydomonas reinhardtii,Synechococcusspecies, andSynechocystisspecies after site-directed mutagenesis and were associated with decreased binding of PS II inhibitors. To our knowledge, this is the first report of a mutation of thepsbAgene at Phe255in a field-selected, herbicide-resistant plant.

Short-term antioxidative responses of 15 microalgae exposed to excessive irradiance including ultraviolet radiation

Short-term photosensitivity and oxidative stress responses were compared for three groups of marine microalgae: Antarctic microalgae, temperate diatoms and temperate flagellates. In total, 15 low-light-acclimated species were exposed to simulated surface irradiance including ultraviolet radiation (SSI). Photosensitivity was assessed as the rate of recovery of Fv/Fm in the hours following SSI treatment. Before, during and after the SSI treatment, oxidative stress responses were assessed by following xanthophyll content and cycling, and activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase, and glutathione redox status. When acclimated to low irradiance, antioxidant levels were not group specific. Superoxide dismutase activity was positively correlated with cell size, whereas in general, ascorbate peroxidase activity appeared to be lower and glutathione redox status appeared to be higher in the Antarctic than in the temperate species. After SSI exposure, the strong inhibition of PSII was followed by variable rates of recovery, although four species remained photosynthetically inactive. SSI tolerance appeared unrelated to geographic or taxonomic background, or to cell size. PSII recovery was enhanced in species with decreasing superoxide dismutase activity, glutathione redox status and increased xanthophyll cycle activity. We conclude that antioxidant responses are highly species specific and not related to the geographic or taxonomic background. Furthermore, xanthophyll cycling seems more important than antioxidants. Finally, it can be hypothesized that glutathione could function as a stress sensor and response regulator.

Physiological Modes of Action of Fluoxetine and its Human Metabolites in Algae

Fluoxetine, the active ingredient of many antidepressants, was identified as specifically toxic toward algae in a quantitative structure-activity relationship (QSAR) analysis with literature data for algae, daphnia, and fish. The goal of this study was to elucidate the mode of action in algae and to evaluate the toxicity of the major human metabolites of fluoxetine using two different algae tests. The time dependence and sensitivity of thedifferenteffectendpointsyield information on the physiological mode of action. Baseline toxicity was predicted with QSARs based on measured liposome-water partition coefficients. The ratio of predicted baseline toxicity to experimental toxicity (toxic ratio TR) gives information on the intrinsic potency (extent of specificity of effect). The metabolite p-trifluoromethylphenol was classified to act as baseline toxicant Fluoxetine (TR 60-150) and its pharmacologically active metabolite norfluoxetine (TR 10-80) exhibited specific toxicity. By comparison with reference compounds we conclude that fluoxetine and norfluoxetine have an effect on the energy budget of algal cells since the time pattern of these two compounds is most similar to that observed for norflurazon, but they act less specifically as indicated by lower TR values and the similarity of the effect pattern to baseline toxicants. The mixture toxicity of fluoxetine and its human metabolites norfluoxetine and p-TFMP can be predicted using the model of concentration addition for practical purposes of risk assessment despite small deviations from this model for the specific endpoints like PSII inhibition because the integrative endpoints like growth rate and reproduction in all cases gave agreement with the predictions for concentration addition.

Increase in the rate of photosynthetic linear electron transport in cyanobacteruim Synechocystis sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.

Hydrogen production by photoautotrophic sulfur‐deprived Chlamydomonas reinhardtii pre‐grown and incubated under high light

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE m(-2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 microE m(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE m(-2) s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O(2) content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 microE m(-2) s(-1), cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H(2). It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.

Maximum and effective PSII yields in the cortex of the main stem of young Prunus cerasus trees: effects of seasons and exposure

Seasonal changes in chlorophyll fluorescence parameters of corticular chlorenchyma in the main trunk of Prunus cerasus were followed in the field under ambient temperature and light conditions during bright days. Concomitantly, measurements of periderm light transmittance also allowed the calculation of linear electron transport rates along PSII. Pre-dawn PSII photochemical efficiency was high during late spring, summer and early autumn, but low during winter in the North-facing, permanently shaded, side and extremely low in the South-facing, exposed side. Corresponding mid-day PSII effective yield and linear electron transport rates peaked at late spring and early summer with the exposed side always displaying lower values for effective yield, but higher values for electron transport rate. However, corticular electron transport rates were more than sixfold lower compared to leaves. Non-photochemical quenching was higher in the exposed side throughout the year while peak values appeared at early autumn. Although a photoinhibitory damage during winter can be claimed, we may note that Mediterranean winter temperatures are mild, while the light reaching the trunk photosynthetic tissues is very low (maximum at 30 and 280 μmol m−2 s−1 in the shaded and the exposed side, respectively) to be considered as photoinhibitory. Based on recent findings for the retention of PSI activity and a concomitant inhibition of PSII under low temperatures in leaves, together with an adequate cyclic electron flow found in bark chlorenchyma, we suggest a temperature-dependent adaptive adjustment in the relative rates of PSI over PSII activity, possibly linked to seasonally changing needs for metabolic energy supply.

Elevated growth temperatures reduce the carbon gain of black spruce [Picea mariana (Mill.) B.S.P.

AbstractWe explored the effect of high‐growth temperatures on a dominant North American boreal tree, black spruce [Picea mariana (Mill.) B.S.P.]. In 2004 and 2005, we grew black spruce at either 22 °C/16 °C day/night temperatures [low temperature (LT)] or 30°/24 °C [high temperature (HT)] and determined how temperature affected growth, leaf morphology, photosynthesis, respiration and thermotolerance. HT spruce were 20% shorter, 58% lighter, and had a 58% lower root : shoot ratio than LT trees. Mortality was negligible in the LT treatment, but up to 14% of HT seedlings died by the end of the growing season. HT seedlings had a higher photosynthetic temperature optimum, but net photosynthesis at growth temperatures was 19–35% lower in HT than LT trees. HT seedlings had both a lower apparent maximum ribulose‐1,5‐bisphosphate carboxylation capacity (Vcmax) and a lower apparent maximum electron transport rate (Jmax) than LT trees, indicating reduced allocation to photosynthetic components. Consistently, HT needles had 26% lower leaf nitrogen content than LT needles. At each measurement temperature, HT seedlings had 20–25% lower respiration rates than LT trees; however, this did not compensate for reduced photosynthetic rates at growth temperature, leading to a greater ratio of dark respiration to net carbon dioxide assimilation rate in HT trees. HT needles had 16% lower concentrations of soluble sugars than LT needles, but similar starch content. Growth at high temperatures increased the thermotolerance of black spruce. HT trees showed less PSII inhibition than LT seedlings and no increase in electrolyte leakage when briefly exposed to 40–57 °C. While trees that develop at high temperatures have enhanced tolerance for brief, extreme heat events, the reduction in root allocation indicates that seedlings will be more susceptible to episodic soil drying and less competitive for belowground resources in future climates of the boreal region.

A European biotype of Amaranthus retroflexus cross‐resistant to ALS inhibitors and response to alternative herbicides

SummaryAn acetolactate synthase (ALS)‐resistant Amaranthus retroflexus biotype was collected in a soyabean crop after repeated exposure to imazethapyr and thifensulfuron‐methyl in north‐eastern Italy. Studies were conducted to characterise the resistance status and determine alternative post‐emergence herbicides for controlling this biotype. Whole‐plant bioassay revealed that the GR50 values were 1898‐ and 293‐fold higher than those observed for the biotype susceptible to imazethapyr and imazamox respectively. The biotype also displayed high cross‐resistance to sulfonylureas. Molecular analysis demonstrated that a single nucleotide substitution had occurred in domain B (TGG to TTG at position 574), conferring a change from the amino acid tryptophan to leucine in the resistant biotype. However, herbicides with other modes of action (PSII, 4‐HPPD and PPO inhibitors) provided excellent control. The GR50 ratios for metribuzin, terbuthylazine and mesotrione were close to 1 and treatments with fomesafen gave 100% control of both susceptible and resistant biotypes at the recommended field dose. This study documents the first case of an imidazolinone and ALS‐resistant biotype in European crops and identifies the post‐emergence herbicide options available for managing this troublesome weed in soyabean crops. Alternative management strategies are also discussed.

Chlorophyll a fluorescence as a biomarker for rapid toxicity assessment

Algal growth assays are the most frequently used methods to detect herbicide toxicity in environmental samples; however, these require several days to detect reductions in growth rate with adequate precision. Hence, a need exists for more rapid assays. Two in vivo chlorophyll a fluorescence assays, one using pulse-amplitude modulation (PAM) fluorescence and another based on fluorescence at 684 and 735 nm, detect the effects of photosystem (PS) II inhibitors (atrazine, diuron, and isoproturon) on Selenastrum capricornutum Printz only after 1 h and 30 min, respectively, of incubation. The median growth inhibition (IC50) could be predicted reliably from effects on three PAM parameters--maximal PSII quantum yield (phi(m)), operational quantum yield (phi'(m)), and nonphotochemical quenching--and from measurements of fluorescence at 684 and 735 nm. The effects of the PSI inhibitor paraquat dichloride, were smaller in magnitude and could be detected after a 24-h incubation. These two in vivo chlorophyll a fluorescence assays can thus provide reliable, rapid, and cost-effective tools to screen toxicity caused by PSII inhibitors. Neither of the two fluorescence assays could consistently predict the effects of nonphotosynthetic inhibitors (alachlor, metsulfuron methyl, and diclofop methyl).

Effects of moderate high-temperature stress on photosynthesis in three saplings of the constructive tree species of subtropical forest

During the exposition to moderate high-temperature stress, photosynthetic rates and fluorescence of chlorophyll a were measured with a photosynthetic measurement system (Li-Cor 6400) and leaf chamber fluorometer (Li-Cor6400 LCF), respectively, in leaves of saplings, sun-adapted species ( Schima superba), shade-adapted species ( Cryptocarya concinna), and in mesophytic plant ( Castanopsis hystrix) (42°C). The results showed that moderate high-temperature stress led to a decrease in F v/ F m, namely the primary photochemical quantum efficiency, indicating that moderate high-temperature stress causes a partial inhibition of PSII. It also showed that such an effect was more severe in the shade-adapted plant C. concinna than in the sun-adapted species S. superba. However, except for the sun-grown leaves of C. concinna, the moderate high-temperature stress increased the photosynthetic rate of leaves at high light intensity. Simultaneously, less photoinhibition was found to occur under high-light intensity, suggesting that the capacity of resistant-photoinhibition was stimulated by moderate high-temperature stress. The quantum yield of PSII (ϕ PSII) decreased in the sun-grown leaves of S. superba and C. hystrix but did not show any significant change in leaves of the shade plant C. concinna and shade-grown leaves of sun plant S. superba or the mesophytic plant C. hystrix because they already had a very low ϕ PSII under this condition. Moderate high-temperature stress led to a decrease in ϕ PSII/ϕ CO 2 ratios, an estimate of the quantum requirement for CO 2 assimilation, in the sun plant S. superba and the mesophytic plant C. hystrix because they were associated with the dissipation of a lower fraction of excitation energy. However, no significant changes were found in shade plant C. concinna and in shade-grown leaves of the other examined plants. The effect of moderate high-temperature (42°C) on photosynthesis depends on species and leaf type (sun and shade leaves) in the saplings of subtropical broad-leaved trees.

PsbA mutation (Asn266 to Thr) in Senecio vulgaris L. confers resistance to several PS II‐inhibiting herbicides

DNA sequence analysis of the psbA gene encoding the D1 protein of photosystem II (PS II), the target site of PS II-inhibiting herbicides, identified a point mutation (Asn266 to Thr) in a bromoxynil-resistant Senecio vulgaris L. population collected from peppermint fields in Oregon. Although this mutation has been previously reported in Synechocystis, this is the first report of this particular point mutation in a higher plant exhibiting resistance to PS II-inhibiting herbicides. The resistant population displayed high-level resistance to bromoxynil and terbacil (R/S ratio 10.1 and 9.3, respectively) and low-level resistance to metribuzin and hexazinone (R/S ratio 4.2 and 2.6, respectively) when compared with the susceptible population. However, the population was not resistant to the triazine herbicides atrazine and simazine or to the urea herbicide diuron. A chlorophyll fluorescence assay confirmed the resistance levels and patterns of cross-resistance of the whole-plant studies. The resistant S. vulgaris plants produced fewer seeds. Differences in cross-resistance patterns to PS II-inhibiting herbicides and the difference in fitness cost could be exploited in a weed management program.

The effect of root‐absorbed PSII inhibitors on Kautsky curve parameters in sugar beet

SummaryThe effects of the photosystem II inhibitors metamitron and terbuthylazine on the shape of the Kautsky (chlorophyll fluorescence induction) curve were investigated in sugar beet grown in hydroponic culture. The objective of the study was to trace recovery processes following herbicide injury using Kautsky curve parameters. Metamitron is used for selective weed control in sugar beet because it is metabolized in this crop. In contrast, terbuthylazine is toxic to sugar beet. Two hours after treatment, various fluorescence induction curve parameters, such as maximum quantum efficiency (FV/Fm), the relative changes at the J step (Fvj) and area (the area between the Kautsky curve and maximum fluorescence, Fm), were affected by metamitron at concentration ranges of 70–280 mg active ingredient (a.i.) L−1 in plants treated at the four‐true‐leaf stage. Shortly after herbicide application, Fv/Fm was more affected by the hydrophilic metamitron [log(Kow) = 0.83] than by the lipophilic terbuthylazine [log(Kow) = 3.21], but these differences between compounds were alleviated as metamitron was metabolized and terbuthylazine was not. Terbuthylazine at 1 mg a.i. L−1 affected sugar beet at the four‐ and six‐true‐leaf stages to the same extent, whereas metamitron at a dose of 140 mg a.i. L−1 affected much more at four‐ than at the six‐true‐leaf stage. Sugar beet recovered from metamitron injury even at high doses (140 and 280 mg a.i. L−1). Fluorescence induction curve parameters were similarly affected by terbuthylazine and, although sugar beet recovered from terbuthylazine injury at low doses (<0.2 mg a.i. L−1), the Kautsky curve was irreversibly affected at higher doses (1–10 mg a.i. L−1), leading finally to plant death. Older plants were affected later, and recovered sooner, from both herbicides.

On the mode of action of the phytotoxin (8 R,16 R)-(−)-pyrenophorin

The response of Avena sterilis to the phytotoxic fungal metabolite (8 R,16 R)-(−)-pyrenophorin was studied under illumination. The phytotoxicity of pyrenophorin at 70 μM was expressed as bleaching of leaf sections which was accompanied by increased electrolyte leakage, elevated superoxide dismutase activity, severe lipid peroxidation, rapid loss of photosynthetic pigments, and a decline in total protein. Proteolytic enzyme activity in the treated tissues was not affected and remained at the same low levels as in the untreated. Pyrenophorin exerts its phytotoxicity through a mechanism operating under both light and dark as indicated by electrolyte leakage measurements. The presence of PSII inhibitor diuron (172 μM) did not reverse electrolyte leakage and bleaching caused by pyrenophorin. The results indicate that the oxidative damage triggered in A. sterilis by the phytotoxin is not light dependent and is exerted through a mechanism which involves electron misdirection and generation of reactive oxygen species.

Multiple herbicide resistance in downy brome (Bromus tectorum) and its impact on fitness

Enhanced herbicide metabolism is less common than target site–based herbicide resistance in weeds and often confers resistance to chemically dissimilar herbicides. In a previous study, the mechanism of acetolactate synthase (ALS)-inhibitor resistance in a downy brome biotype was determined to be metabolism. Our research was aimed at determining the multiple resistance pattern in the downy brome biotype, establishing its physiological basis, and investigating its fitness. Dose–response experiments showed that the resistant biotype was also moderately resistant to ethofumesate, clethodim, fluazifop, diuron, and terbacil and highly resistant to the triazine herbicides, atrazine and metribuzin. DNA sequence analysis of the psbA gene, which is the target site of PSII inhibitors, demonstrated a single amino acid substitution from serine to glycine in the resistant biotype at residue 264 in the D1 protein. Thus, the resistant biotype contains two different resistance mechanisms, herbicide metabolism and an altered target site. The resistant biotype produced less shoot dry weight, leaf area, and seed and was shorter than the susceptible biotype. The resistant biotype was also less competitive than the susceptible biotype. Thus, in the absence of herbicides, the frequency of this resistant biotype is unlikely to increase in a population of mixed downy brome biotypes.

Herbicide impact on Hormosira banksii gametes measured by fluorescence and germination bioassays

The innovative bioassay described here involves chlorophyll a fluorescence measurements of gametes from the macroalgae, Hormosira banksii, where gametes (eggs) were exposed to Diuron, Irgarol and Bromacil. Response was assessed as percent inhibition from control of effective quantum yield (Δ F/Fm′) of photosystem II, herein referred to as % PSII Inhibition. This was measured with the dual-channelled pulse amplitude modulated (PAM) fluorometer, ToxY-PAM. The fluorescence bioassay was run simultaneously with an established H. banksii germination bioassay to compare sensitivity, precision, and time-to-result. The fluorescence bioassay gave highly sensitive results evidenced by EC 50s (% PSII Inhibition) for Diuron, Irgarol and Bromacil being three, four and three orders of magnitude (respectively) lower than EC 50s generated from the germination bioassays. Precision of the fluorescence bioassay was demonstrated with low coefficient of variations (<30%) for all three toxicants. With regard to time, the fluorescence bioassay gave results within 6 h, as opposed to more than 50 h for the germination bioassay.

Nutritional and chlorophyll fluorescence responses of lucerne (Medicago sativa) to waterlogging and subsequent recovery

Periodic flooding of perennial crops such as lucerne (Medicago sativa,L) is a major cause of lowered productivity and leads in extreme cases to plant death. In this study, effects of waterlogging and subsequent recovery on plant nutrient composition and PSII photochemistry were studied to gain a better understanding of the mechanisms of recovery as they relate to leaf photochemistry (chlorophyll fluorescence) and nutrient dynamics. Three lucerne cultivars and one breeding line were flooded for 20 d, drained and left to recover for another 16 d under glasshouse conditions. Leaf and root nutrient composition (P, K, Ca, Mg, B, Cu and Zn) of waterlogged lucerne was significantly lower than in freely drained controls, leaf N concentrations were also significantly lower in waterlogged lucerne. At the same time, there were significantly (5-fold) higher concentrations of Fe in waterlogged roots and Na in leaves (2-fold) of stressed plants. PS II photochemistry, which was impaired due to waterlogging, recovered almost fully after 16 d of free drainage in all genotypes. Alongside fluorescence recovery, concentrations of several nutrients also increased in recovered plants. Growth parameters, however, remained suppressed after draining. The latter was due to both the smaller capacity of CO2 assimilation in previously waterlogged plants (caused in part by nutrient deficiency and associated inhibition of PSII) and the plant’s need to re-direct available nutrient and assimilate pools to repair the damage to the photosynthetic apparatus and roots. It is concluded, that for any lucerne-breeding program it is important to determine not only the degree of tolerance to waterlogging but also the potential for recovery of different genotypes, as well as look for ‘outstanding individuals’ within each population.

Does the effect of herbicide pulse exposure on aquatic plants depend on Kow or mode of action?

The highest concentrations of herbicides measured in flowing surface waters are often only present for short periods of time. These herbicide pulses can reach concentrations that would affect aquatic plants if present over a long time. The aim of this study was to assess the effect of a 3-h herbicide pulse relative to the effects of long-term (4 and 7 days) exposure of six herbicides with different sites of action and different K ow on the growth of the floating macrophyte Lemna minor. The herbicides were the two photosynthetic inhibitors: diquat and terbuthylazine, the inhibitors of acetolactate syntase (ALS), imazamox and metsulfuron-methyl and the microtubule assembly inhibitors propyzamide and pendimethalin. The log K ow ranged from −4.6 to 5.2. For imazamox, metsulfuron-methyl, propyzamide and pendimethalin a 3-h pulse induced the effect on area-specific growth as did a 4-day exposure at an approximate 10-fold higher concentration. For diquat and terbuthylazine a concentration closer to a factor of 100 or more was needed for a 3-h pulse to induce an effect similar to that of a 4-day exposure. For diquat, the low pulse-effect was most likely due to a slow uptake of the hydrophilic ion (log K ow = −4.6), as no effect was observed on chlorophyll fluorescence within 8 h after exposure. The chlorophyll fluorescence parameters are expected to respond quickly to a PSI inhibitor as diquat. For terbuthylazine, fluorescence measurements showed an effect on photosynthesis within 1 h of exposure, and reached a minimum after 3 h. Recovery was fast, and initial fluorescence was restored within 24 h. Hence, the small pulse effect on area-specific growth was due to rapid recovery of photosynthesis. In contrast to terbuthylazine, the stop in area-specific growth observed for the ALS-and microtubule assembly inhibitors, took up to 4 days to recover from. Such a long recovery time after a pulse of only 3 h indicate that at realistic pulse exposures of up to a day or two, pulse-effects will approach the effects obtained in long-term studies. When investigating the effects of pulse exposures on aquatic plants, we should therefore focus more on non-photosynthetic inhibitors, which might not appear in pulses in as large concentrations as the PSII inhibitors investigated up till now, but whose effect, even in a shorter pulse, can be more damaging.

The effects of a PSII inhibitor on phytoplankton community structure as assessed by HPLC pigment analyses, microscopy and flow cytometry

Measurements of the stress imposed by a PSII inhibiting herbicide (Irgarol 1051 ®) on the composition of a phytoplankton community was investigated by comparing chemotaxonomy, as determined by high performance liquid chromatography (HPLC), optical microscopy and analytical flow cytometry (AFC). Changes in community structure were induced in microcosms containing a natural marine phytoplankton community exposed to different concentrations of Irgarol 1051 ® (0.5 and 1.0 μg l −1). Microcosms were maintained under controlled laboratory conditions in semi-continuous culture over 120 h. Class-specific phytoplankton biomass (chlorophyll a) was estimated using CHEMTAX analyses of pigment concentrations. Microscopic identification and carbon content estimates were cross-correlated with CHEMTAX and also with AFC enumeration/size classifications of major phytoplankton groups. CHEMTAX–HPLC analyses and microscopy results demonstrated that prasinophytes and prymnesiophytes were the most affected groups following exposure to Irgarol 1051 ®. The selective reductions in both classes as estimated by both techniques revealed similar trends. Results for chlorophytes and dinoflagellates showed these groups to be most tolerant to Irgarol 1051 ®. Indeed, class-specific biomass for chlorophytes as determined by CHEMTAX and microscopy were correlated ( R 2 = 0.53) which demonstrated an increase in both abundance and carbon content following exposures to Irgarol 1051 ®. Abundances of nanoeukaryotes as determined by microscopy afforded good agreement with results from AFC ( R 2 = 0.8), although for picoeukaryotes, abundances were underestimated by microscopy ( R 2 = 0.43). The relative performance of the selected techniques is discussed.