Changes in the growth rate of Chlamydomonas reinhardtii under long-term selection by temperature and salinity: Acclimation vs. evolution.

We investigated the roles of acclimation and different components involved in evolution (adaptation, chance and history) on the changes in the growth rate of the model freshwater microalga Chlamydomonas reinhardtii P. A. Dang. exposed to selective temperature and salinity. Three C. reinhardtii strains previously grown during one year in freshwater medium and 20 °C were exposed to 5 °C temperature increase and a salinity of 5 g L-1 NaCl. Cultures under each selective scenario and in combination (increase of salinity and temperature), were propagated until growth rate achieved an invariant mean value for 6 months (100-350 generations, varying as a function of scenario and strain). The changes of the growth rate under increased temperature were due to both adaptation and acclimation, as well as history. However, acclimation was the only mechanism detected under salinity increase as well as in the selective scenario of both temperature and salinity, suggesting that genetic variability would not allow survival at salinity higher than that to which experimental populations were exposed. Therefore, it could be hypothesized that under a global change scenario an increase in salinity would be a greater challenge than warming for some freshwater phytoplankton.

Photosynthetic performance in cyanobacteria with increased sulphide tolerance: an analysis comparing wild-type and experimentally derived strains.

Sulphide is proposed to have influenced the evolution of primary stages of oxygenic photosynthesis in cyanobacteria. However, sulphide is toxic to most of the species of this phylum, except for some sulphide-tolerant species showing various sulphide-resistance mechanisms. In a previous study, we found that this tolerance can be induced by environmental sulphidic conditions, in which two experimentally derived strains with an enhanced tolerance to sulphide were obtained from Microcystis aeruginosa, a sensitive species, and Oscillatoria, a sulphide-tolerant genus. We have now analysed the photosynthetic performance of the wild-type and derived strains in the presence of sulphide to shed light on the characteristics underlying the increased tolerance. We checked whether the sulphide tolerance was a result of higher PSII sulphide resistance and/or the induction of sulphide-dependent anoxygenic photosynthesis. We observed that growth, maximum quantum yield, maximum electron transport rate and photosynthetic efficiency in the presence of sulphide were less affected in the derived strains compared to their wild-type counterparts. Nevertheless, in 14C photoincoporation assays, neither Oscillatoria nor M. aeruginosa exhibited anoxygenic photosynthesis using sulphide as an electron donor. On the other hand, the content of photosynthetic pigments in the derived strains was different to that observed in the wild-type strains. Thus, an enhanced PSII sulphide resistance appears to be behind the increased sulphide tolerance displayed by the experimentally derived strains, as observed in most natural sulphide-tolerant cyanobacterial strains. However, other changes in the photosynthetic machinery cannot be excluded.

Detection of the maximum resistance to the herbicides diuron and glyphosate, and evaluation of its phenotypic cost, in freshwater phytoplankton.

One of the most important anthropogenic impacts on freshwater aquatic ecosystems close to intensive agriculture areas is the cumulative increase in herbicide concentrations. The threat is especially relevant for phytoplankton organisms because they have the same physiological targets as the plants for which herbicides have been designed. This led us to explore the evolutionary response of three phytoplanktonic species to increasing concentrations of two herbicides and its consequences in terms of growth and photosynthesis performance. Specifically, we used an experimental ratchet protocol to investigate the differential evolution and the limit of resistance of a cyanobacterium (Microcystis aeruginosa) and two chlorophyceans (Chlamydomonas reinhardtii and Dictyosphaerium chlorelloides) to two herbicides in worldwide use: glyphosate and diuron. Initially, the growth rate of M. aeruginosa and D. chlorelloides was completely inhibited when they were exposed to a dose of 0.23 ppm diuron or 40 ppm glyphosate, whereas a higher concentration of both herbicides (0.46 ppm diuron or 90 ppm glyphosate) was necessary to abolish C. reinhardtii growth. However, after running a ratchet protocol, the resistance of the three species to both herbicides increased by an adaptation process. M. aeruginosa and D. chlorelloides were able to grow at 1.84 ppm diuron and 80 ppm glyphosate and C. reinhardtii proliferated at twice these concentrations. Herbicide-resistant strains showed lower growth rates than their wild-type counterparts in the absence of herbicides, as well as changes on morphology and differences on photosynthetic pigment content. Besides, herbicide-resistant cells generally showed a lower photosynthetic performance than wild-type strains in the three species. These results indicate that the introduction of both herbicides in freshwater ecosystems could produce a diminution of primary production due to the selection of herbicide-resistant mutants, that would exhibit lower photosynthetic performance than wild-type populations.

Adaptation dynamics and evolutionary rescue under sulfide selection in cyanobacteria: a comparative study between Microcystis aeruginosa and Oscillatoria sp. (cyanobacteria).

Experimental evolution studies using cyanobacteria as model organisms are scarce despite the role of cyanobacteria in the evolution of photosynthesis. Three different experimental evolution approaches have been applied to shed light on the sulfide adaptation process, which played a key role in the evolution of this group. We used a Microcystis aeruginosa sulfide-sensitive strain, unable to grow above ~0.1 mM, and an Oscillatoria sp. strain, isolated from a sulfureous spa (~0.2 mM total sulfide). First, performing a fluctuation analysis design using the spa waters as selective agent, we proved that M. aeruginosa was able to adapt to this sulfide level by rare spontaneous mutations. Second, applying a ratchet protocol, we tested if the limit of adaptation to sulfide of the two taxa was dependent on their initial sulfide tolerance, finding that M. aeruginosa adapted to 0.4 mM sulfide, and Oscillatoria sp. to ~2 mM sulfide, twice it highest tolerance level. Third, using an evolutionary rescue approach, we observed that both speed of exposure to increasing sulfide concentrations (deterioration rate) and populations' genetic variation determined the survival of M. aeruginosa at lethal sulfide levels, with a higher dependence on genetic diversity. In conclusion, sulfide adaptation of sensitive cyanobacterial strains is possible by rare spontaneous mutations and the adaptation limits depend on the sulfide level present in strain's original habitat. The high genetic diversity of a sulfide-sensitive strain, even at fast environmental deterioration rates, could increase its possibility of survival even to a severe sulfide stress.

Direct uptake of HCO3- in the marine angiosperm Posidonia oceanica (L.) Delile driven by a plasma membrane H+ economy.

Seagrasses access HCO3- for photosynthesis by 2 mechanisms, apoplastic carbonic anhydrase-mediated dehydration of HCO3- to CO2 and direct HCO3- uptake. Here, we have studied plasma membrane energization and the mechanism for HCO3- import in Posidonia oceanica. Classical electrophysiology and ion-selective microelectrodes were used to measure the membrane potential, cytosolic pH, and the cytosolic concentrations of Na+ and Cl- upon the addition of HCO3- . The photosynthetic response to HCO3- and to inhibitors was also measured. Results indicate that the primary pump of P. oceanica plasma membrane is a fusicoccin-sensitive H+ -ATPase. Bicarbonate depolarizes the plasma membrane voltage and transiently acidifies the cytosol, indicating that HCO3- is transported into the cells by an H+ -symport. Initial cytosolic acidification is followed by an alkalinization, suggesting an internal dehydration of HCO3- . The lack of cytosolic Na+ and Cl- responses rules out the contribution of these ions to HCO3- transport. The energetics of nH+ /HCO3- symport allows, for n = 1, an estimate of cytosolic accumulation of 0.22 mM HCO3- . Because this transporter could permit accumulation of HCO3- up to 100 times above the equilibrium concentration, it would be a significant component of a carbon-concentrating mechanism in this species.

A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions.

The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H(+) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H(+) efflux (H(+)-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H(+)-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H(+) efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K(+) efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H(+)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H(+)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H(+)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.

The limit of the genetic adaptation to copper in freshwater phytoplankton.

Copper is one of the most frequently used algaecides to control blooms of toxic cyanobacteria in water supply reservoirs. Among the negative impacts derived from the use of this substance is the increasing resistance of cyanobacteria to copper toxicity, as well as changes in the community structure of native phytoplankton. Here, we used the ratchet protocol to investigate the differential evolution and maximum adaptation capacity of selected freshwater phytoplankton species to the exposure of increasing doses of copper. Initially, a dose of 2.5 µM CuSO4·5H2O was able to completely inhibit growth in three strains of the toxic cyanobacterium Microcystis aeruginosa, whereas growth of the chlorophyceans Dictyosphaerium chlorelloides and Desmodesmus intermedius (represented by two different strains) was completely abolished at 12 µM. A significant increase in resistance was achieved in all derived populations during the ratchet experiment. All the chlorophyceans were able to adapt to up to 270 µM of copper sulfate, but 10 µM was the highest concentration that M. aeruginosa strains were able to cope with, although one of the replicates adapted to 30 µM. The recurrent use and increasing doses of copper in water reservoirs could lead to the selection of copper-resistant mutants of both chlorophyceans and cyanobacteria. However, under high concentrations of copper, the composition of phytoplankton community could undergo a drastic change with cyanobacteria being replaced by copper-resistant chlorophyceans. This result stems from a distinct evolutionary potential of these species to adapt to this substance.

A dominant-negative form of Arabidopsis AP-3 ß-adaptin improves intracellular pH homeostasis.

Intracellular pH (pHi ) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pHi regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid-tolerant 1-1D (wat1-1D) are due to the expression of a truncated form of AP-3 ß-adaptin (encoded by the PAT2 gene) that behaves as a as dominant-negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pHi and a more depolarized plasma membrane electrical potential than wild-type cells. Additional phenotypes of wat1-1D roots include increased rates of acetate efflux, K(+) uptake and H(+) efflux, the latter reflecting the in vivo activity of the plasma membrane H(+) -ATPase. The in vitro activity of the enzyme was not increased but, as the H(+) -ATPase is electrogenic, the increased ion permeability would allow a higher rate of H(+) efflux. The AP-3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1-1D mutant can be explained if loss of function of the AP-3 ß-adaptin causes activation of channels or transporters for organic anions (acetate) and for K(+) at the plasma membrane, perhaps through miss-localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.

Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis.

Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H(+) -ATPase) is indirect: over-expression of ROF2 activates K(+) uptake, causing depolarization of the plasma membrane, which activates the electrogenic H(+) pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.

An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface.

A genetic screen of Arabidopsis 'activation-tagging' mutant collection based on tolerance to norspermidine resulted in a dominant mutant (par1-1D) with increased expression of the QSO2 gene (At1g15020), encoding a member of the quiescin-sulfhydryl oxidase (QSO) family. The par1-1D mutant and transgenic plants overexpressing QSO2 cDNA grow better than wild-type Arabidopsis in media with toxic cations (polyamines, Li(+) and Na(+)) or reduced K(+) concentrations. This correlates with a decrease in the accumulation of toxic cations and an increase in the accumulation of K(+) in xylem sap and shoots. Conversely, three independent loss-of-function mutants of QSO2 exhibit phenotypes opposite to those of par1-1D. QSO2 is mostly expressed in roots and is upregulated by K(+) starvation. A QSO2Colon, two colonsGFP fusion ectopically expressed in leaf epidermis localized at the cell wall. The recombinant QSO2 protein, produced in yeast in secreted form, exhibits disulfhydryl oxidase activity. A plausible mechanism of QSO2 action consists on the activation of root systems loading K(+) into xylem, but different from the SKOR channel, which is not required for QSO2 action. These results uncover QSOs as novel regulators of ion homeostasis.

Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance.

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.

Biochemical and physiological responses of Gracilaria tenuistipitata uuder two different nitrogen treatments.

The influence of nitrogen on ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco. EC 4.1.1.39) content is poorly understood in macroalgae. N-deficient Gracilaria tenuistipitata Zhang et Xia var. liui was cultivated in the laboratory under constant light intensity and temperature. Biochemical and physiological variables were monitored after a high (1 mM) or low (o. 1 mM) nitrate pulse. Rubisco content in crude extracts was estimated by SDS-PAGE with the Coomassie Blue Staining procedure. Nitrate was consumed immediately in the low-N treatment, but there was always an external nitrate source in the high-N treatment. Total soluble proteins and phycobiliproteins decreased as internal nitrogen diminished in the low-N treatment, but kept fairly constant in N-sufficient conditions. However, Rubisco content increased until the 7th day and then started to decrease in both cases. Fresh weight increment showed a better correlation with Rubisco than with pigment content.