Preconditioning of cortical neurons by oxygen-glucose deprivation: tolerance induction through abbreviated neurotoxic signaling

Transient exposure of rat cortical cultures to nonlethal oxygen-glucose deprivation (OGD preconditioning) induces tolerance to otherwise lethal oxygen-glucose deprivation (OGD) or N-methyl-D-aspartate 24 h later. This study evaluates the role of cytosolic and mitochondrial Ca²⁺-dependent cellular signaling. Mechanistic findings are placed in context with other models of ischemic preconditioning or known neurotoxic pathways within cortical neurons. Tolerance to otherwise lethal OGD is suppressed by performing OGD preconditioning in the presence of the broad-scope catalytic antioxidants Mn(III)tetra(4-carboxyphenyl)porphyrin (MnTBAP) or Zn(II)tetra(4-carboxyphenyl)porphyrin [Zn(II)TBAP], but not by a less active analog, Mn(III)tetra(4-sulfonatophenyl)porphyrin, or a potent superoxide scavenger, Mn(III)tetra(N-ethyl-2-pyridyl)porphyrin chloride. Inhibitors of adenosine A₁ receptors, nitric oxide synthase, mitogen-activated protein kinase, and poly(ADP-ribose) polymerase fail to suppress OGD preconditioning despite possible links with reactive oxygen species in other models of ischemic preconditioning. Preconditioning is suppressed by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which has been ascribed elsewhere to inhibition of superoxide transport to the cytosol through mitochondrial anion channels. However, although it induces mitochondrial Ca²⁺ uptake, neuronal preconditioning is largely insensitive to mitochondrial uncoupling with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone or 2,4-dinitrophenol. Un-couplers will prevent production of mitochondrial reactive oxygen species, implying nonmitochondrial targets by MnTBAP, Zn(II)TBAP, and DIDS. Emphasizing the importance of an increase in cytosolic Ca²⁺ during preconditioning, a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, KN-62, suppresses development of subsequent tolerance. Summarizing, only those cellular transduction pathways that have the potential to be neurotoxic may be activated by preconditioning in cortical neurons. Finally, a marked decrease in extracellular glutamate is observed during otherwise lethal OGD in preconditioned cultures, suggesting that this end effector may represent a point of convergence across different preconditioning models. N-methyl-D-aspartate; Ca²⁺; antioxidants; mitochondria     

Expression of antioxidant enzymes in human inflammatory cells

Because antioxidant enzymesmay have an important role in the oxidant resistance ofinflammatory cells, we investigated the mRNA levels and specificactivities of manganese and copper-zinc superoxide dismutases (Mn SODand Cu,Zn SOD), catalase (Cat), and glutathione peroxidase, as well asthe concentrations of glutathione (GSH) in human neutrophils,monocytes, monocyte-derived macrophages, and alveolar macrophages.Levels of GSH and glutathione peroxidase activity in monocytes werethree times higher than in neutrophils, whereas the mRNA of Cat was50-fold and its specific activity 4-fold higher in neutrophils.Although Mn SOD mRNA levels were higher in neutrophils, enzymeactivities, as well as those of Cu,Zn SOD, were similar in allphagocytic cells. Neutrophils lost their viability, assessed by adeninenucleotide depletion, within 24 h ex vivo and more rapidly if GSH wasdepleted. However, neutrophils were the most resistant cell type toexogenousH₂O₂.In conclusion, high Cat activity of neutrophils appears to explaintheir high resistance against exogenousH₂O₂,whereas low GSH content and GSH-related enzymes seem to account for thepoor survival of human neutrophils.

     

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation,the expression of TcMT2a, TcMT2b, and TcMT3 was analysed inthree accessions and 15 F₃ families of two inter-accession crossesof the Cd/Zn hyperaccumulator Thlaspi caerulescens, with differentdegrees of Zn accumulation. The highest expression levels werefound in the shoots of a superior metal-accumulating calamineaccession from St Laurent le Minier, with >10-fold TcMT3expression compared with another calamine accession and a non-metallicolousaccession. Moreover, F₃ sibling lines from the inter-accessioncrosses that harboured the MT2a or MT3 allele from St Laurentle Minier had higher expression levels. However, there was noco-segregation of TcMT2a or TcMT3 expression and Zn accumulation.To examine the functions of TcMTs in plants, TcMT2a and TcMT3were ectopically expressed in Arabidopsis. The transformantlines had reduced root length in control medium but not at highmetal concentrations, suggesting that the ectopically expressedproteins interfered with the physiological availability of essentialmetals under limited supply. The Arabidopsis transformant linesdid not show increased tolerance to Cd, Cu, or Zn, nor increasedCd or Zn accumulation. Immunohistochemical analysis indicatedthat in roots, MT2 protein is localized in the epidermis androot hairs of both T. caerulescens and Arabidopsis thaliana.The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarilyinvolved in Zn accumulation as such. However, the elevated expressionlevels in the metallicolous accessions suggests that they docontribute to the metal-adapted phenotype, possibly throughimproving Cu homeostasis at high Zn and Cd body burdens. Alternatively,they might function as hypostatic enhancers of Zn or Cd tolerance. Key words: Cd, crosses, metallothionein, protein, quantitative real-time PCR, Thlaspi caerulescens, Zn Received 14 August 2008; Revised 14 October 2008 Accepted 15 October 2008     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

Adenine nucleotide-creatine-phosphate module in myocardial metabolic system explains fast phase of dynamic regulation of oxidative phosphorylation

Computational models of a large metabolic system can be assembled from modules that represent a biological function emerging from interaction of a small subset of molecules. A "skeleton model" is tested here for a module that regulates the first phase of dynamic adaptation of oxidative phosphorylation (OxPhos) to demand in heart muscle cells. The model contains only diffusion, mitochondrial outer membrane (MOM) permeation, and two isoforms of creatine kinase (CK), in cytosol and mitochondrial intermembrane space (IMS), respectively. The communication with two neighboring modules occurs via stimulation of mitochondrial ATP production by ADP and P_i from the IMS and via time-varying cytosolic ATP hydrolysis during contraction. Assuming normal cytosolic diffusion and high MOM permeability for ADP, the response time of OxPhos (t_mito; generalized time constant) to steps in cardiac pacing rate is predicted to be 2.4 s. In contrast, with low MOM permeability, t_mito is predicted to be 15 s. An optimized MOM permeability of 21 µm/s gives t_mito = 3.7 s, in agreement with experiments on rabbit heart with blocked glycolytic ATP synthesis. The model correctly predicts a lower t_mito if CK activity is reduced by 98%. Among others, the following predictions result from the model analysis: 1) CK activity buffers large ADP oscillations; 2) ATP production is pulsatile in beating heart, although it adapts slowly to demand with "time constant" 14 heartbeats; 3) if the muscle isoform of CK is overexpressed, OxPhos reacts slower to changing workload; and 4) if mitochondrial CK is overexpressed, OxPhos reacts faster. systems biology; computational model; creatine kinase; phosphocreatine shuttle; regulatory module; mitochondrial membrane permeability; oxygen consumption     

Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G₂/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G₂/M arrest and growth retardation. amyotrophic lateral sclerosis; copper; zinc superoxide dismutase; G₂/M arrest; neurodegenerative disease     

Nutrient Dilution by Starch in CO2-enriched Chrysanthemum

Increasing growth irradiance and CO₂ generally decreases foliarnutrient concentration on a dry weight basis and increases foliarstarch concentration. However, the extent to which starch concentrations‘dilute’ foliar nutrient concentrations when thelatter are expressed on a dry weight basis is not known. Todetermine the importance of differential starch accumulationin calculating nutrient concentrations on a dry weight basis,leaf nutrient and starch concentrations were measured in Chrysanthemum? morifolium ‘Fiesta’ (Ramat.) cuttings grown atthree irradiance levels and two CO₂ levels for eight weeks inboth winter and spring. On a dry weight basis, foliar concentrationsof most nutrients were lower in both seasons as a result ofthe elevated CO₂ and irradiance levels, and total dry weightswere higher. Per cent starch was greater at the high CO₂ levelin both seasons but was only greater at higher irradiances inthe winter experiment. When starch was subtracted from the leafdry weights, the differences between CO₂ and irradiance treatmentsdisappeared with respect to N, P, K, Ca, Mg, S, and B but notfor Fe, Mn, Zn, and Cu. Key words: CO₂ enrichment, starch, nutrients, irradiance     

Structural organization, regulation, and expression of the chloroplastic superoxide dismutase Sod1 gene in maize.

A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined.     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

Plant Characteristics and Nutrient Composition and Mobility of Broccoli (Brassica oleracea var. italica) Supplied with NH4+, NO3"" or NH4NO3

Shelp, B. J. 1987. Plant characteristics and nutrient compositionand mobility of broccoli (Brassica oleracea var. italica) suppliedwith NH⁺₄, NO₃ or NH₄NO₃.—J. exp. Bot. 38: 1603–1618. The effects of varying NH⁺₄, NO₃ or NH₄NO₃ concentration onthe final plant characteristics, element composition, and accumulationof NO₃-N, NH⁺₄-N and organic-N were evaluated in broccoli (Brassicaoleracea var. italicacv. Futura and Premium Crop) plants culturedin vermiculite under greenhouse conditions supplemented withlight. NH⁺₄-grown plants were stunted and exhibited signs ofmarginal necrosis on the old leaves, accompanied by an accumulationof NH4. The tissue levels of N, P, Mn, Cu, Zn and B were generallyincreased by NH⁺₄ versus NO₃ nutrition whereas the reverse wastrue for Ca; Mg and K were only slightly affected, if at all.These results are attributed to: changes in element availabilityresulting from reduced rhizosphere pH due to NH⁺₄uptake ratherthan NO ₃uptake; competition of Ca uptake by NH⁺₄; and dilutionof N by increased vegetative growth with NO₃-nutrition. Theelement concentrations of N, P or K were similar in all tissueswhereas Ca, B and Mn were markedly less in the florets and youngleaves compared to mature leaves; this supports literature indicatingthat the former elements are phloem-mobile whereas the latterare not. Assuming that the nutrient supply for mature leavesis delivered principally via the xylem stream, the data suggestthat nutrients for developing leaves and florets are suppliedpredominantly in the phloem. If so, under our experimental conditions.Zn and Cu were also readily mobile in the phloem whereas Mgmovement was restricted. NH₄⁺ versus NO₄⁺ J nutrition alteredthe distribution of these elements. The two broccoli cultivarstested under the greenhouse environment differed in NH⁺₄ toleranceand in the distribution of K and Cu suggesting there was a geneticbasis for cultivar variation in mineral acquisition and redistribution. Key words: Plant nutrition, phloem mobility, elemental composition.     

Selection of Copper and Zinc Resistant Nicotiana plumbaginifolia Cell Suspension Cultures

Copper and zinc resistant cells of Nicotiana plumbaginifoliawere selected using unmutagenized cell suspensions in mediumcontaining normally lethal concentrations of CuSO₄ or ZnSO₄.Both resistances were retained for thirty cell doublings withoutselection pressure. The Cu resistant cells were 10-times andthe Zn resistant cells were 6-times as resistant as the wildtype cells. The Zn resistant cells were also somewhat resistantto AlCl₃ in comparison with the wild type cells, while the Curesistant cells were also somewhat resistant to ZnSO₄ and AlCl₃.The uptake of Cu by the Cu resistant cells and Zn by the Znresistant cells was higher than that of the wild type cells. (Received April 21, 1986; Accepted June 30, 1986)     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation. mitochondria; apoptosis; necrosis, O-linked-N-acetylglucosamine     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Regulation of Mineral Redistribution in Pod-Bearing Soybean Explants

On the way from the roots to the seeds during reproductive developmentin soybean (Glycine max), a large proportion of the mineralspass through the leaves rather than travelling directly viathe xylem. This direct and indirect movement of mineral nutrientshas important implications for mineral redistribution, seeddevelopment and leaf senescence. Therefore, we have studiedthe role of cytokinin and mineral flux from the roots in regulatingmineral redistribution from the leaves to the seeds using explants,i.e. a leaf, a pod and a subtending stem segment, with theirbases immersed in treatment solutions. Thus, defined solutionscontaining cytokinin and/or minerals can be substituted forthe roots. When explants (excised at early-mid podfill) aresupplied H₂O only, leaf N, P, K, Mo, Mg, Zn, Fe, B, Cu, Ca,and Mn decline, ranging from 93% for Mo to 38% for Fe. In explantson H₂O, N, P, K, Mo, Mg, Zn, and Fe appear to be redistributedfrom the leaves to the seeds, while the B, Cu, Ca, and Mn lostfrom the leaves do not seem to move to the seeds. Although amixture of minerals resembling xylem sap can delay net lossof these elements from the leaves, it does not prevent the decreases.The cytokinin zeatin (4.6 µM) inhibits the loss of N,IC, Mo, Mg, Zn, Fe, B, Cu, Ca, and Mn from the leaves, but notthat of P. When combined with minerals, zeatin not only preventsthe loss of the minerals from the leaves but may even greatlyincrease them with the possible exception of Zn, Fe, and Cu.Supplying the mineral nutrient mixture increases the quantitiesof N, P, K, Mg, Cu, and B in the seeds but not Zn, Fe, Mn, Ca,and Mo. For those minerals, especially N, where zeatin inhibitsefflux from the leaves, it may reduce the amounts in the seeds,but it does not change P, K, Mg, and Ca. The accumulation andredistribution patterns of the different mineral nutrients showmany dissimilarities thereby suggesting differences in the controlof their distribution. Key words: Cytokinin, mineral transport, seed development, senescence     

Regulation of Photosystem Stoichiometry in the Photosynthetic System of the Cyanophyte Synechocystis PCC 6714 in Response to Light-Intensity

Light-induced changes in stoichiometry among three thylakoidcomponents, PS I, PS II and Cyt b₆-f complexes, were studiedwith the cyanophyte Synechocystis PCC 6714. Special attentionwas paid to two aspects of the stoichiometric change; first,a comparison of the patterns of regulation in response to differencesin light-intensity with those induced by differences in light-quality,and second, the relationship between regulation of the stoichiometryand the steady state of the electron transport system. Resultsfor the former indicated that (1) the abundance of PS I on aper cell basis was reduced under white light at the intensityas high as that for light-saturation of photosynthesis, butPS I per cell was increased under low light-intensity, (2) PSII and Cyt b₆-f complexes remained fairly constant, and (3)changes in the abundance of PS I depended strictly on proteinsynthesis. The pattern was identical with that of chromaticregulation. For the second problem, the redox steady-statesof Cyt f and P700 under white light of various intensities weredetermined by flash-spectroscopy. Results indicated that (1)Cyt f and P700 in cells grown under low light-intensity [highratio of PS I to PS II (PS I/PS II)] were markedly oxidizedwhen the cells were exposed to high light-intensity, while theyremained in the reduced state under low light-intensity. (2)After a decrease in the abundance of PS I, most of P700 remainedin the reduced state even under high light-intensity, whilethe level of reduced Cyt f remained low. (3) Both Cyt f andP700 in cells of low PS I/PS II were fully reduced under lowlight-intensity, and Cyt f reduction following the flash wasrapid, which indicates that the turnover of PS I limits theoverall rate of electron flow. After an increase in the abundanceof PS I, the electron transport recovered from the biased state.(4) The redox steady-state of the Cyt b₆-f complex correlatedwell with the regulation of PS I/PS II while the state of thePQ pool did not. Based on these results, a working model ofthe regulation of assembly of the PS I complex, in which theredox steady-state of the Cyt b₆-f complex is closely relatedto the primary signal, is proposed. (Received August 2, 1990; Accepted December 10, 1990)     

Genetic analysis of an electrophoretic variant for the chloroplast-associated form of Cu/Zn superoxide dismutase in sunflower (Helianthus annuus L.)

Polyacrylamide gel electrophoresis of water-soluble proteinsfrom sunflower (Helianthus annuus L.) cotyledons, followed byspecific staining for superoxide dismutase activity, discriminated,according to their electrophoretic mobility, two distinct achromaticbands for Cu/Zn superoxide dismutase. Zymograms of proteinsfrom isolated chloroplasts showed that the chloroplast-locatedCu/Zn superoxide dismutase (Cu/ZnSOD_Chl) migrated faster inthe SOD activity-stained gels. An electrophoretic variant pattern,whose mobility is lower than the control pattern, was identifiedin the ABA-deficient mutant w-1. The variant is coded by a nucleargene with two codominant alleles. Key words: Sunflower, Helianthus annuus L., ABA-deficient mutant, electrophoretic isozyme variant, superoxide dismutase     

Variations in the Mineral Composition of Herbarium Plant Species Collected During the Last Three Centuries

Mineral content (dry weight basis) was determined for herbariumspecimens of 12 C₃ plants (trees, shrubs and herbs) collectedduring the last 250 years in N.E. Spain. Present values of Al,Ca, Cu, Sr, Fe, P, Mg, Mn, K, Na, S, and Zn were always lowerthan in any other period of the last three centuries. Only oneC₄ plant was analysed. It presented a similar pattern to theC₃ plants. These results are in accordance with experimentalresults that have shown that the mineral content of plants grownin elevated CO₂ is generally lowered. Increased atmosphericCO₂ and other anthropogenic environmental changes are suggestedas possible causes of the changes in mineral content. Key words: Leaf mineral content, Al, Ca, Cu, Sr, Fe, P, Mg, Mn, K, Na, S, Zn, herbaria, last three centuries