Cadmium Accumulation Involves Synthesis of Glutathione and Phytochelatins, and Activation of CDPK, CaMK, CBLPK, and MAPK Signaling Pathways in Ulva compressa

In order to analyze the effect of cadmium in Ulva compressa (Chlorophyta), the alga was cultivated with 10, 25, and 50 μM of cadmium for 7 days, and the level of intracellular cadmium was determined. Intracellular cadmium showed an increase on day 1, no change until day 5, and an increase on day 7. Then, the alga was cultivated with 10 μM for 7 days, and the level of hydrogen peroxide, superoxide anions, and lipoperoxides; activities of antioxidant enzymes ascorbate peroxidase (AP), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); the level of glutathione (GSH) and ascorbate (ASC); and the level of phytochelatins (PCs) and transcripts encoding metallothioneins (UcMTs) levels were determined. The level of hydrogen peroxide increased at 2 and 12 h, superoxide anions on day 1, and lipoperoxides on days 3 to 5. The activities of AP and GR were increased, but not the DHAR activity. The level of GSH increased on day 1, decreased on day 3, and increased again on day 5, whereas ASC slightly increased on days 3 and 7, and activities of enzymes involved in GSH and ASC synthesis were increased on days 3 to 7. The level of PC2 and PC4 decreased on day 3 but increased again on day 5. The level of transcripts encoding UcMT1 and UcMT2 increased on days 3 to 5, mainly that of UcMT2. Thus, cadmium accumulation induced an oxidative stress condition that was mitigated by the activation of antioxidant enzymes and synthesis of GSH and ASC. Then, the alga cultivated with inhibitors of calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calcineurin B-like protein kinases (CBLPKs), and MAPKs and 10 μM of cadmium for 5 days showed a decrease in intracellular cadmium and in the level of GSH and PCs, with the four inhibitors, and in the level of transcripts encoding UcMTs, with two inhibitors. Thus, CDPKs, CaMK, CBLPKS, and MAPKs are involved in cadmium accumulation and GSH and PC synthesis, and GSH and PCs and/or UcMTs may participate in cadmium accumulation

Ulva compressa from Copper-Polluted Sites Exhibits Intracellular Copper Accumulation, Increased Expression of Metallothioneins and Copper-Containing Nanoparticles in Chloroplasts

In order to analyze the mechanisms involved in copper accumulation in Ulva compressa, algae were collected at control sites of central and northern Chile, and at two copper-polluted sites of northern Chile. The level of intracellular copper, reduced glutathione (GSH), phytochelatins (PCs), PC2 and PC4, and transcripts encoding metallothioneins (MTs) of U. compressa, UcMT1, UcMT2 and UcMT3, were determined. Algae of control sites contained around 20 μg of copper g−1 of dry tissue (DT) whereas algae of copper-polluted sites contained 260 and 272 μg of copper g−1 of DT. Algae of control sites and copper-polluted sites did not show detectable amounts of GSH, the level of PC2 did not change among sites whereas PC4 was increased in one of the copper-polluted sites. The level of transcripts of UcMT1 and UcMT2 were increased in algae of copper-polluted sites, but the level of UcMT3 did not change. Algae of a control site and a copper-polluted site were visualized by transmission electron microscopy (TEM) and the existence of copper in electrodense particles was analyzed using energy dispersive x-ray spectroscopy (EDXS). Algae of copper-polluted sites showed electrodense nanoparticles containing copper in the chloroplasts, whereas algae of control sites did not. Algae of a control site, Cachagua, were cultivated without copper (control) and with 10 μM copper for 5 days and they were analyzed by TEM-EDXS. Algae cultivated with copper showed copper-containing nanoparticles in the chloroplast whereas control algae did not. Thus, U. compressa from copper-polluted sites exhibits intracellular copper accumulation, an increase in the level of PC4 and expression of UcMTs, and the accumulation of copper-containing particles in chloroplasts.This work was financed by Fondecyt Regular 1160013 to A.M and by Dicyt-USACH

Daily changes on seasonal ecophysiological responses of the intertidal brown macroalga Lessonia spicata: Implications of climate change

Global climate change is expected to have detrimental effects on coastal ecosystems, with impacts observable at the local and regional levels, depending on factors such as light, temperature, and nutrients. Shifts in dominance between primary producers that can capitalize on carbon availability for photosynthesis will have knock-on effects on marine ecosystems, affecting their ecophysiological responses and biological processes. Here, we study the ecophysiological vulnerability, photoacclimation capacity, and tolerance responses as ecophysiological responses of the intertidal kelp Lessonia spicata (Phaeophyceae, Laminariales) during a year through different seasons (autumn, winter, spring, and summer) in the Pacific Ocean (central Chile). Six different daily cycle experiments were carried out within each season. A battery of different biochemical assays associated with antioxidant responses and in-vivo chlorophyll a fluorescence parameter showed that during spring and summer, there was an increase in photosynthetic capacity in the macroalgae, although their responses varied depending on light and nutrient availability in the course of the year. Lessonia spicata showed maximal photosynthesis and a similar photoinhibition pattern in summer compared to the other seasons, and the contents of nitrate and phosphorous in seawater were less in winter. Thus, high irradiance during spring and summer displayed a higher maximal electron transport rate (ETRmax), irradiance of saturation (Ek), non-photochemical quenching (NPQmax), nitrogen and carbon contents, and photoprotector compound levels. Antioxidant activity increased also in summer, the seasonal period with the highest oxidative stress conditions, i.e., the highest level of hydrogen peroxide (H2O2). In contrast, under low irradiance, i.e., wintertime conditions, L. spicata demonstrated lower concentrations of the photosynthetic pigments such as chlorophyll a and carotenoids. Our study suggests that macroalgae that are subjected to increased irradiance and water temperature under lower nutrient availability mediated by seasonal changes (expected to worsen under climate change) respond with higher values of productivity, pigment contents, and photoprotective compounds. Thus, our findings strengthen the available evidence to predict that algae in the order Laminariales, specifically L. spicata (kelp), could better proliferate, with lower vulnerability and greater acclimation, than other marine species subject to future expected conditions associated with climate change.Financial and logistical support was granted by the project of Fondo Nacional de Desarrollo Científico y Tecnológico, Chile through grant Project FONDECYT, Chile N° 11180197, ANID, Chile - provided to Paula Celis - Plá

Plant growth-promoting rhizobacteria and root system functioning

The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture

Microbial diversity in soils suppressive to Fusarium diseases

Fusarium species are cosmopolitan soil phytopathogens from the division Ascomycota, which produce mycotoxins and cause significant economic losses of crop plants. However, soils suppressive to Fusarium diseases are known to occur, and recent knowledge on microbial diversity in these soils has shed new lights on phytoprotection effects. In this review, we synthesize current knowledge on soils suppressive to Fusarium diseases and the role of their rhizosphere microbiota in phytoprotection. This is an important issue, as disease does not develop significantly in suppressive soils even though pathogenic Fusarium and susceptible host plant are present, and weather conditions are suitable for disease. Soils suppressive to Fusarium diseases are documented in different regions of the world. They contain biocontrol microorganisms, which act by inducing plants’ resistance to the pathogen, competing with or inhibiting the pathogen, or parasitizing the pathogen. In particular, some of the Bacillus, Pseudomonas, Paenibacillus and Streptomyces species are involved in plant protection from Fusarium diseases. Besides specific bacterial populations involved in disease suppression, next-generation sequencing and ecological networks have largely contributed to the understanding of microbial communities in soils suppressive or not to Fusarium diseases, revealing different microbial community patterns and differences for a notable number of taxa, according to the Fusarium pathosystem, the host plant and the origin of the soil. Agricultural practices can significantly influence soil suppressiveness to Fusarium diseases by influencing soil microbiota ecology. Research on microbial modes of action and diversity in suppressive soils should help guide the development of effective farming practices for Fusarium disease management in sustainable agriculture

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.</jats:p

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis

There is currently no information regarding the role that whole mitogen activated protein kinase (MAPK) pathways play in counteracting environmental stress in photosynthetic organisms. To address this gap, we exposed Ulva compressa to chronic levels of copper (10 µM) specific inhibitors of Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK), and Cytokinin Specific Binding Protein (p38) MAPKs alone or in combination. Intracellular copper accumulation and photosynthetic activity (in vivo chlorophyll a fluorescence) were measured after 6 h, 24 h, 48 h, and 6 days of exposure. By day 6, when one (except JNK) or more of the MAPK pathways were inhibited under copper stress, there was a decrease in copper accumulation compared with algae exposed to copper alone. When at least two MAPKs were blocked, there was a decrease in photosynthetic activity expressed in lower productivity (ETRmax), efficiency (αETR), and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax), compared to both the control and copper-only treatments. In terms of accumulation, once the MAPK pathways were partially or completely blocked under copper, there was crosstalk between these and other signaling mechanisms to enhance metal extrusion/exclusion from cells. Crosstalk occurred among MAPK pathways to maintain photosynthesis homeostasis, demonstrating the importance of the signaling pathways for physiological performance. This study is complemented by a parallel/complementary article Rodríguez-Rojas et al. on the role of MAPKs in copper-detoxification.</jats:p

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction