Exogenous Mn2 Research Articles

Calcium Rescues Streptococcus pneumoniae D39 ΔmntE Manganese-Sensitive Growth Phenotype.

Calcium (Ca2+) functions as a universal signal messenger in eukaryotes but in bacteria, the physiological roles for Ca2+ are limited. Here, we examine the role of Ca2+ in Streptococcus pneumoniae during manganese (Mn2+) intoxication. S. pneumoniae mntE mutants, lacking the Mn2+ efflux transporter, exhibit impaired growth due to accumulation of Mn2+ when exposed to elevated exogenous Mn2+. This Mn2+-sensitive growth defect is restored to wild-type growth level by exogenous Ca2+, in a Ca2+-dependent manner. Despite growth restoration of the mntE mutant to wild-type levels, cellular Mn2+ remains elevated in this strain. Bacterial capsule production is also increased for the mntE mutant, resulting in reduced adherence capacity to surfaces and poor biofilm formation, which is consistent with it experiencing Mn2+ intoxication. Ca2+ presence did not significantly impact bacterial capsule production or biofilm formation. Further analysis of the cell morphology demonstrates that Ca2+ contributes to cell division and reduces cell chain lengths. Together, these data describe the first role of Ca in S. pneumoniae that has potential implications in bacterial virulence since Ca affects cell division and likely Mn2+-associated cellular processes.

Target-driven cascade amplified assembly of covalent organic frameworks on tetrahedral DNA nanostructure with multiplex recognition domains for ultrasensitive detection of microRNA

BackgroundMicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis. ResultsIn this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the “freeze-drying-reduction” method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn2+. Without the addition of exogenous Mn2+, the dynamically-generated Mn2+-powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM. Significance and noveltyTaking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn2+-powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening.

Mineralization of Ni2+-Bearing Mn Oxide through Simultaneous Sequestration of Ni2+ and Mn2+ by Enzymatically Active Fungal Mn Oxides

A fungus, Acremonium strictum KR21-2, produces biogenic manganese oxides (BMOs) that can oxidize exogenous Mn2+ ions to form different BMO phases. When other guest ions are present during the BMO formation, it can strongly affect the mineralogical characteristics of the resultant BMO phase. The impact of coexisting Ni2+ ions on the mineralogy of BMO phases formed through enzymatic Mn(II) oxidation and its sequestration ability is not yet fully understood. To better understand it, repeated sequestration experiments were conducted using BMOs in Ni2+/Mn2+ binary, single Ni2, and single Mn2+ solution systems with a pH range of 6.0 to 7.5. It was observed that simultaneous sequestration of Ni2+ and Mn2+ was efficient, with irreversible Ni2+ incorporation at pH values above 7.0. The resultant BMO phases showed that Ni2+-bearing Mn oxides resembling feitknechitite (β-MnOOH) were developed through enzymatic Mn(II) oxidation. At pH values below 6.5, the turbostratic birnessite structure was maintained even in Ni2+/Mn2+ binary solutions, and subsequently, the Ni2+ sequestration efficiency was low. The pseudo-first-order rate constants of enzymatically inactivated BMOs for Mn2+ sequestration were two orders of magnitude lower than those of active BMOs, indicating the crucial role of the enzymes in precipitating Ni2+-bearing Mn oxide phases. These findings provide new insights into the mechanism of Ni2+ interaction with Mn oxide through microbial activity under circumneutral pH conditions.

The Development of Aspergillus flavus and Biosynthesis of Aflatoxin B1 are Regulated by the Golgi-Localized Mn2+ Transporter Pmr1.

Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn2+) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis- and medial-Golgi compartments was critical in facilitating Mn2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-N-acetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.

Effect of Osmolytes on Photoassembly of Functionally Active Mn4CaO5 Cluster in Mn-Depleted Photosystem II Preparations Isolated from Spinach Leaves

The effect of osmolytes (trehalose, sucrose, betaine, D-glucose and hydroxyectoine) on the photoassembly of the functionally active inorganic core of the water-oxidizing complex (Mn4CaO5 cluster) in Mn-depleted PSII preparations (apo-WOC-PSII) was investigated. It was revealed that the efficiency of the photoassembly of the Mn4CaO5 cluster was considerably (three times) increased in the presence of 1 M disaccharides (trehalose or sucrose) in contrast to other osmolytes. It was found that the osmolytes (especially trehalose or sucrose) improved the redox interaction of exogenous Mn2+ with apo-WOC-PSII, enhanced the protective effect of Mn2+ against the photoinhibition of apo-WOC-PSII, protected CaCl2-treated PSII preparations against thermoinactivation, and stabilized the water-oxidizing complex and electron transport from QA to QB in native PSII preparations during heat treatment. It is suggested that the ability of osmolytes to enhance the efficiency of the photoassembly of a Mn4CaO5 cluster depends on their effect on the following key processes: the redox interaction of Mn2+ with apo-WOC-PSII; the stability of apo-WOC-PSII to photoinhibition during the photoactivation procedure; and the stabilization of both the newly assembled functionally active Mn4CaO5 cluster and the electron transfer from QA to QB.

Development of a DNAzyme Walker for the Detection of APE1 in Living Cancer Cells.

DNAzyme walker technology is a compelling option for bioanalytical and drug delivery applications. While nucleic acid and protein targets have been used to activate DNAzyme walkers, investigations into enzyme-triggered DNAzyme walkers in living cells are still in their early stages. The base excision repair (BER) pathway presents an array of enzymes that are overexpressed in cancer cells. Here, we introduce a DNAzyme walker system that sensitively and specifically detects the BER enzyme apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1). We constructed the DNAzyme walker on the surface of 20 nm-diameter gold nanoparticles. We achieved a detection limit of 160 fM of APE1 in a buffer and in whole cell lysate equivalent to the amount of APE1 in a single HeLa cell in a sample volume of 100 μL. Confocal imaging of the DNAzyme walking reveals a cytoplasmic distribution of APE1 in HeLa cells. Walking activity is tunable to exogenous Mn2+ concentrations and the uptake of the DNAzyme walker system does not require transfection assistance. We demonstrate the investigative potential of the DNAzyme walker for up-regulated or overactive enzyme biomarkers of the BER pathway in cancer cells.

Calcium disodium edetate controls citrus green mold by chelating Mn2+ and targeting pyruvate synthesis in the pathogen

Edetate salts are a kind of food additive regarded as candidates for chemical fungicides because of their lower toxicity and higher safety for the environment and human health. The antifungal activities of edetate salts have been determined in some phytopathogens except Penicillium digitatum, which causes the most destructive fungal disease during the citrus postharvest period. In the present study, we investigated the molecular mechanisms of calcium disodium edetate (EDTA-Na2Ca) against P. digitatum in deepth. The results suggested that EDTA-Na2Ca inhibited the P. digitatum P44 strain in vitro at a minimum inhibitory/fungicidal concentration (MIC/MFC) of 50 mmol L−1. Meanwhile, EDTA-Na2Ca also inhibited P. digitatum in vivo by enhancing the host defense system. The P44 strain treated with EDTA-Na2Ca displayed a delayed conidial germination rate, expanded hyphal tips, thickened septa and cell walls, and destroyed mitochondria. Transcriptome sequencing and qRT-PCR analyses indicated that EDTA-Na2Ca suppressed the P44 strain primarily by disturbing the carbohydrate metabolism pathway, especially the synthesis of pyruvate in glycolysis. In addition, the adverse effects, including those on mycelial growth, pyruvate synthesis and superoxide dismutase activity, of EDTA-Na2Ca could be reversed by exogenous Mn2+, implying that Mn2+ chelation is an essential mode of action. In summary, the current work verifies that EDTA-Na2Ca is a potential antifungal drug that cures citrus green mold probably by chelating Mn2+ and targeting pyruvate synthesis in P. digitatum.

Sequestration of Oxyanions of V(V), Mo(VI), and W(VI) Enhanced through Enzymatic Formation of Fungal Manganese Oxides

Biogenic Mn oxides (BMOs) have become captivating with regard to elemental sequestration, especially at circumneutral pH conditions. The interaction of BMOs with oxyanions, such as vanadate (V), molybdate (VI), and tungstate (VI), remains uncertain. This study examined the sequestration of V(V), Mo(VI), and W(VI) (up to ~1 mM) by BMOs formed by the Mn(II)-oxidizing fungus, Acremonium strictum KR21-2. When A. strictum KR21-2 was incubated in liquid cultures containing either Mo(VI) or W(VI) with soluble Mn2+, the oxyanions were sequestered in parallel with enzymatic Mn(II) oxidation with the maximum capacities of 8.8 mol% and 28.8 mol% (relative to solid Mn), respectively. More than 200 μM V(V) showed an inhibitory effect on growth and Mn(II) oxidizing ability. Sequestration experiments using preformed primary BMOs that maintained the enzymatic Mn(II) oxidizing activity, with and without exogenous Mn2+, demonstrated the ongoing BMO deposition in the presence of absorbent oxyanions provided a higher sequestration capacity than the preformed BMOs. X-ray diffraction displayed a larger decline of the peak arising from (001) basal reflection of turbostratic birnessite with increasing sequestration capacity. The results presented herein increase our understanding of the role of ongoing BMO formation in sequestration processes for oxyanion species at circumneutral pH conditions.

Biodegradable MnO-Based Nanoparticles with Engineering Surface for Tumor Therapy: Simultaneous Fenton-Like Ion Delivery and Immune Activation.

Immune checkpoint inhibitors have achieved significant clinical success but are still suffering from inadequate immune activation. It is worth noting that manganese as a nutritional inorganic trace element is closely associated with immune activation to fight against tumor growth and metastasis via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. Herein, we designed hollow mesoporous silica-coated MnO nanoparticles (NPs), followed by conjugation of tumor homing peptide iRGD (CRGDKGPD). The obtained NPs (MnO@mSiO2-iRGD NPs) were applied to magnetic resonance imaging (MRI)-guided tumor immune-chemodynamic combination therapy, in which MnO NPs can be harnessed for cGAS-STING pathway-activated immunotherapy, Fenton-like reaction-induced reactive oxygen species upregulation, and T1-weighted MRI. The rough surface and large cavities of the mSiO2 shell promote cellular uptake and MnO NPs delivery. Meanwhile, it was found that MnO@mSiO2-iRGD NPs would dissociate under an acid environment, resulting in tumor specificity of MRI and exogenous Mn2+ release. Our results revealed that these pH-responsive biodegradable MnO@mSiO2-iRGD NPs synergized with α-PD-1 (PD-1 = programmed cell death-1) blocking antibody to highly elicit cytotoxic T lymphocyte infiltration and restrict melanoma progression and metastasis, which were envisioned as a promising candidate for tumor theranostics.

Simultaneous Sequestration of Co2+ and Mn2+ by Fungal Manganese Oxide through Asbolane Formation

Biogenic manganese oxides (BMOs) have attractive environmental applications owing to their metal sequestration and oxidizing abilities. Although Co readily accumulates into Mn oxide phases in natural environments, the Co2+ sequestration process that accompanies the enzymatic Mn(II) oxidation of exogenous Mn2+ remains unknown. Therefore, we prepared newly formed BMOs in a liquid culture of Acremonium strictum KR21-2 and conducted repeated sequestration experiments in a Mn2+/Co2+ binary solution at pH 7.0. The sequestration of Co2+ by newly formed BMOs (~1 mM Mn) readily progressed in parallel with the oxidation of exogenous Mn2+, with higher efficiencies than that in single Co2+ solutions when the initial Co2+ concentrations (0.16–0.8 mM) were comparable to or lower than the exogenous Mn2+ concentration (~0.8 mM). This demonstrates a synergetic effect on Co sequestration. Powder X-ray diffraction showed a typical pattern for asbolane only when newly formed BMOs were treated in Mn2+/Co2+ binary systems, implying that the enzymatic Mn(II) oxidation by newly formed BMOs favored asbolane formation. Cobalt K-edge X-ray absorption near-edge structure measurements showed that both Co(II) and Co(III) participated in the formation of the asbolane phase in the binary solutions, whereas most of the primary Co2+ was sequestered as Co(III) in the single Co2+ solutions, which partly explains the synergetic effects on Co sequestration efficiency in the binary solutions. The results presented here provide new insights into the mechanism of Co interaction with Mn oxide phases through asbolane formation by enzymatic Mn(II) oxidation under circumneutral pH conditions.

Preferential Elimination of Ba2+ through Irreversible Biogenic Manganese Oxide Sequestration

Biogenic manganese oxides (BMOs) formed in a culture of the Mn(II)-oxidizing fungus Acremonium strictum strain KR21-2 are known to retain enzymatic Mn(II) oxidation activity. Consequently, these are increasingly attracting attention as a substrate for eliminating toxic elements from contaminated wastewaters. In this study, we examined the Ba2+ sequestration potential of enzymatically active BMOs with and without exogenous Mn2+. The BMOs readily oxidized exogenous Mn2+ to produce another BMO phase, and subsequently sequestered Ba2+ at a pH of 7.0, with irreversible Ba2+ sequestration as the dominant pathway. Extended X-ray absorption fine structure spectroscopy and X-ray diffraction analyses demonstrated alteration from turbostratic to tightly stacked birnessite through possible Ba2+ incorporation into the interlayer. The irreversible sequestration of Sr2+, Ca2+, and Mg2+ was insignificant, and the turbostratic birnessite structure was preserved. Results from competitive sequestration experiments revealed that the BMOs favored Ba2+ over Sr2+, Ca2+, and Mg2+. These results explain the preferential accumulation of Ba2+ in natural Mn oxide phases produced by microbes under circumneutral environmental conditions. These findings highlight the potential for applying enzymatically active BMOs for eliminating Ba2+ from contaminated wastewaters.

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt photooxidation by PSII reaction center (demonstrated at the first time), (2) Cyt photoreduction via O2−• photoproduced on the acceptor side of PSII, and (3) Cyt photoreduction by reduced electron carriers of PSII. A step-by-step removal of components of water-oxidizing complex was accompanied by the appearance of Cyt photooxidation, an increase in the superoxide dismutase (SOD)-dependent Cyt photoreduction (related to O2−• formation), and a decrease in the SOD-independent Cyt photoreduction. Re-addition of PsbO protein diminished the Cyt-induced restoration of electron transfer in PSII. Addition of diuron led to inhibition of these photoprocesses, while exogenous Mn2+ inhibited only the Cyt c photooxidation. The results can be important for correct measurements of O2−• photoproduction in PSII and for elucidation of the role of cytochrome c550 in cyanobacterial PSII.

Sequestration of La3+ by fungal manganese oxides and the effect of Mn(II) oxidase activity

This study examined the sequestration of La3+ by biogenic manganese oxides (BMOs), formed by the Mn(II)-oxidizing fungus Acremonium strictum strain KR21-2. When newly formed BMOs were added to La(NO3)3 solutions under aerobic conditions at pH 6.0, effective sequestration of La3+ (23.5 mol% relative to Mn oxide) was observed without any release of Mn2+. On the other hand, under anaerobic conditions the BMOs released significant amounts of Mn2+ and exhibited lower sequestration efficiencies, especially at low La3+ concentrations. The reason is that La3+ sequestration by BMOs is accompanied by the release of Mn2+ due to ion exchange, and the released Mn2+ competes with La3+ for sorption on the surface. The Mn(II) oxidase in the newly formed BMOs can progressively oxidize Mn2+ and thereby enhance La3+ sequestration. Repeated treatments of newly formed BMOs demonstrated that enzymatic Mn(II) oxidation readily converted exogenous Mn2+ to Mn oxide, providing new sites for sequestering La3+. The addition of NaN3 deactivated the associated enzyme and subsequently released La3+ from the BMO surface due to sorption competition by exogenous Mn2+. Therefore, the Mn(II) oxidase activity is important for maintaining high La3+ sequestration efficiency of the BMOs. Experiments with mixed solutions indicated that the BMOs preferentially sequester La3+ and Cd2+ over Zn2+ and Ni2+. These results provide new insights on the recovery of La3+ (and possibly other trivalent rare earth ions) through the BMOs.

Enhancement of photoassembly of the functionally active water-oxidizing complex in Mn-depleted photosystem II membranes upon transition to anaerobic conditions

It has been shown earlier (Khorobrykh and Klimov, 2015) that molecular oxygen is directly involved in the general mechanism of the donor side photoinhibition of photosystem II (PSII) membranes. In the present work the effect of oxygen on photoassembly (“photoactivation”) of the functionally active inorganic core of the water-oxidizing complex (WOC) in Mn-depleted PSII preparations (apo-WOC-PSII) in the presence of exogenous Mn2+, Ca2+ as well as ferricyanide was investigated. It was revealed that the efficiency of the photoassembly of the WOC was considerably increased upon removal of oxygen from the medium during photoactivation procedure using the enzymatic oxygen trap or argon flow. The lowering of O2 concentration from 250μM to 75μM, 10μM and near 0μM results in 29%, 71% and 92%, respectively, stimulation of the rate of O2 evolution measured after the photoactivation. The increase in the intensity of light used during the photoactivation was accompanied by a decrease of both the efficiency of photoassembly of the WOC and the stimulation effect of removal of O2 (that may be due to the enhancement of the processes leading to the photodamage to PSII). It is concluded that the enhancement in photoactivation of oxygen-evolving activity of apo-WOC-PSII induced by oxygen removal from the medium is due to the suppression of the donor side photoinhibition of PSII in which molecular oxygen can be involved.

Photoinhibition of photosynthesis in water deficit leaves of grapevine (Vitis vinifera L.) plants

Photoinhibition under irradiance of 2 000 µmol m−2 s−1 (HI) was studied in detached control (C) and water deficit (WD) leaves of grapevine (Vitis vinifera L.) plants. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. The potential efficiency of photosystem (PS) 2, Fv/Fm, marginally declined under HI in WD-leaves without significant increase of F0. In contrast, Fv/Fm ratio declined markedly with significant increase of F0 in C-leaves. In isolated thylakoids, the rate of whole chain and PS2 activity under HI were more decreased in C-than WD-leaves. The artificial exogenous electron donors diphenyl carbazide, NH2OH, and Mn2+ failed to restore the HI-induced loss of PS2 activity in both C-and WD-leaves. Thus HI operates at the acceptor side of PS2 in both leaf types. Quantification of the PS2 reaction centre protein D1 following HI exposure of leaves showed pronounced differences between C-and WD-leaves. The marked loss of PS2 activity under HI of C-leaves was due to the marked loss of D1 protein of the PS2 reaction centre.

Involvement of tannase in the acquisition of manganese from tannin-rich medium by tannase-producing Lactobacillus plantarum

The role of tannase produced by Lactobacillus plantarum in tannin-rich medium was demonstrated by using strains with known tannase activities. After 20 h incubation in aerobic tannin-rich medium, the growth of L. plantarum KOG 19 with low activity was lower than that of L. plantarum ATCC 14917T with high activity by approximately 1.5 orders of magnitude, whereas the growth of KOG 19 in aerobic medium without tannin was almost the same as that of ATCC 14917T. The reduced growth of KOG 19 in aerobic tannin-rich medium was recovered by the addition of exogenous tannase or Mn2+ to the medium. A similar result was obtained in the comparison of growth levels between high and low tannase activity groups in aerobic tannin-rich medium, while no significant differences were observed between the groups in anaerobic tannin-rich medium. Furthermore, the growth of the low tannase activity group in aerobic tannin-rich medium was restored by the addition of catalase. These results suggest that tannase production allows L. plantarum strains to acquire and accumulate Mn2+ which is otherwise chelated by tannins, and then high intracellular Mn2+ compensates for the lack of the enzyme superoxide dismutase in L. plantarum resulting in resistance to oxygen toxicity under aerobic conditions.

Photoinhibition of photosynthesis in leaves of grapevine (Vitis vinifera L. cv. Riesling). Effect of chilling nights

Photoinhibition of photosynthesis was investigated in control (C) and chilling night (CN) leaves of grapevine under natural photoperiod at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) and photosynthetic electron transport measurements. When the potential efficiency of photosystem (PS) 2, Fv/Fm was measured at midday, it markedly declined with significant increase of F0 in CN leaves. In isolated thylakoids, the rate of whole chain and PS2 activity were markedly decreased in CN leaves than control leaves at midday. A smaller inhibition of PS1 activity was also observed in both leaf types. Later, the leaves reached maximum PS2 efficiencies similar to those observed in the morning during sampling at evening. The artificial exogenous electron donors diphenyl carbazide, NH2OH, and Mn2+ failed to restore the PS2 activity in both leaf types at midday. Thus CN enhanced inactivation on the acceptor side of PS2 in grapevine leaves. Quantification of the PS2 reaction centre protein D1 following midday exposure of leaves showed pronounced differences between C and CN leaves. The marked loss of PS2 activity in CN leaves noticed in midday samples was mainly due to the marked loss of D1 protein of the PS2 reaction centre.

High Irradiance Induced Changes of Photosystem 2 in Young and Mature Needles of Cypress (Cupressus sempervirens L.)

Photoinhibition of photosynthesis was studied in young and mature detached sun needles of cypress under high irradiance (HI) of about 1 900 μmol m-2 s-1. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. Compared with the mature needles, the young needles, containing about half the amount of Chl a+b per unit area, exhibited a higher proportion of total carotenoids (Car) as xanthophyll cycle pigments and had an increased ratio of Car/Chl a+b. The potential efficiency of photosystem (PS) 2, Fv/Fm, markedly declined in HI-treated young needles without significant increase of F0 level. In contrast, the Fv/Fm ratio declined with significant increase of F0 level in mature needles. In isolated thylakoids, the rate of whole chain and PS2 activity markedly decreased in young HI-needles in comparison with mature needles. A smaller inhibition of PS1 activity was observed in both needles. In the subsequent dark incubation, fast recovery was found in both needle Types that reached maximum PS2 efficiencies similar to those observed in non-photoinhibited needles. The artificial exogenous electron donors DPC, NH2OH, and Mn2+ failed to restore the HI-induced loss of PS2 activity in mature needles, while DPC and NH2OH significantly restored it in young needles. Hence, HI-inactivation was on the donor side of PS2 in young needles and on the acceptor side of PS2 in mature needles. Quantification of the PS2 reaction centre proteins D1 and 33 kDa protein of water splitting complex following HI-exposure of needles showed pronounced differences between young and mature needles. The large loss of PS2 activity in HI-needles was due to the marked loss of D1 protein of the PS2 reaction centre in mature needles and of the 33 kDa protein in young needles.

Novel promoter sequence required for manganese regulation of manganese peroxidase isozyme 1 gene expression in Phanerochaete chrysosporium.

Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3' end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism.

Photoinhibition of Photosynthesis in Sun and Shade Grown Leaves of Grapevine (Vitis vinifera L.)

The degree of photoinhibition of sun and shade grown leaves of grapevine was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. The potential efficiency of photosystem 2 (PS2), Fv/Fm, markedly declined under high irradiance (HI) in shade leaves with less than 10 % of F0 level. In contrast, Fv/Fm ratio declined with about 20 % increase of F0 level in sun leaves. In isolated thylakoids, the rate of whole chain and PS2 activity in HI shade and sun leaves was decreased by about 60 and 40 %, respectively. A smaller inhibition of photosystem 1 (PS1) activity was also observed in both leaf types. In the subsequent dark incubation, fast recovery was observed in both leaf types that reached maximum PS2 efficiencies similar to non-photoinhibited control leaves. The artificial exogenous electron donors DPC, NH2OH, and Mn2+ failed to restore the HI-induced loss of PS2 activity in sun leaves, while DPC and NH2OH were significantly restored in shade leaves. Hence HI in shade leaves inactivates on the donor side of PS2 whereas it does at the acceptor side in sun leaves, respectively. Quantification of the PS2 reaction centre protein D1 and the 33 kDa protein of water splitting complex following HI-treatment of leaves showed pronounced differences between shade and sun leaves. The marked loss of PS2 activity in HI leaves was due to the marked loss of D1 protein of the PS2 reaction centre protein and the 33 kDa protein of the water splitting complex in sun and shade leaves, respectively.