Influence of Water and Temperature Stress on the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The temperature dependence of the rate and magnitude of the reappearance of photosystem II (PSII) variable fluorescence following illumination has been used to determine plant temperature optima. The present study was designed to determine the effect of a plant's environmental history on the thermal dependency of the reappearance of PSII variable fluorescence. In addition, this study further evaluated the usefulness of this fluorescence technique in identifying plant temperature optima. Laboratory and greenhouse grown potato (Solanum tuberosum L. cv “Norgold M”) plants had a thermal kinetic window between 15 and 25°C. The minimum apparent K_m of NADH hydroxypyruvate reductase for NADH occurred at 20°C. This temperature was also the temperature providing maximal reappearance of variable fluorescence. Soybean (Glycine max [L.] Merrill cv “Wayne”) plants had a thermal kinetic window between 15 and 30°C with a minimum apparent K_m at 25°C. Maximal reappearance of variable fluorescence was seen between 20 and 30°C. To determine if increasing environmental temperatures increased the temperature optimum provided from the fluorescence response curves, potato and soybean leaves from irrigated and dryland field grown plants were evaluated. Although the absolute levels of PSII variable fluorescence declined with increasing thermal stress, the temperature optimum of the dryland plants did not increase with increased exposure to elevated temperatures. Because of variability in the daily period of high temperature stress in the field, studies were initiated with tobacco plants grown in controlled environment chambers. The reappearance of PSII variable fluorescence in tobacco (Nicotiana tabacum L. cv “Wisconsin 38”) leaves that had experienced continuous leaf temperatures of 35°C for 8 days had the same 20°C optima as leaves from plants grown at room temperature. The results of this study suggest that the temperature optimum for the reappearance of variable fluorescence following illumination is not altered by the plant's previous exposure to variable environmental temperatures. These findings support the usefulness of this procedure for the rapid identification of a plant's temperature optimum.     

Temporal Phases in Apoptosis Defined by the Actions of Src Homology 2 Domains, Ceramide, Bcl-2, Interleukin-1β Converting Enzyme Family Proteases, and a Dense Membrane Fraction

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10–100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60–90 min of the process (the “initiation” phase). Next, Bcl-2, interleukin-1β converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring ~90–120 min after the start of incubation (the “sentencing” phase). In the final phase (“execution”), the process of active nuclear destruction ensues.     

Flow cytometry of spinach chloroplasts : determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right-side-out vesicles of the thylakoid membranes.     

Effect of Water Extracts of Carob Pods, Tannic Acid, and Their Derivatives on the Morphology and Growth of Microorganisms

The effect of aqueous extracts of carob (Ceratonia siliqua) pods, gallotannic acid, gallic acid, and catechol on several microorganisms was studied. Carob pod extract and tannic acid showed a strong antimicrobial activity toward some cellulolytic bacteria. On the basis of tannin content, to which antimicrobial effect was related, carob pod extracts inhibited Cellvibrio fulvus and Clostridium cellulosolvens at 15 μg/ml, Sporocytophaga myxococcoides at 45 μg/ml, and Bacillus subtilis at 75 μg/ml. The inhibiting concentrations for tannic acid were found to be 12, 10, 45, and 30 μg/ml, respectively. Gallic acid and catechol were much less effective. Tannic acid and the tannin fraction of carob extract exerted both bacteriostatic and bactericidal effects on C. fulvus. Respiration of C. fulvus in the presence of bactericidal concentrations of tannic acid or tannin fraction of carob extract was inhibited less than 30%. A partial formation of “protoplasts” by C. fulvus was obtained after 2 hr of incubation in a growth medium to which 20% sucrose, 0.15% MgSO₄·7H₂O, and 10 to 50 μg/ml of tannic acid or 500μg/ml of penicillin, or both, had been added. Tannic acid and the tannin fraction of carob extract protected C. fulvus from metabolic lysis in sucrose solution. Although the growth of other microorganisms tested was only slightly affected, the morphology of some of them was drastically changed in the presence of subinhibitory concentrations of carob pod extracts of tannic acid. It is suggested that the site of action of tannins on sensitive microorganisms is primarily the cell envelope.     

Uncovering a Microbial Enigma: Isolation and Characterization of the Streamer-Generating,Iron-Oxidizing,Acidophilic Bacterium “Ferrovum myxofaciens”

A betaproteobacterium, shown by molecular techniques to have widespread global distribution in extremely acidic (pH 2 to 4) ferruginous mine waters and also to be a major component of “acid streamer” growths in mine-impacted water bodies, has proven to be recalcitrant to enrichment and isolation. A modified “overlay” solid medium was devised and used to isolate this bacterium from a number of mine water samples. The physiological and phylogenetic characteristics of a pure culture of an isolate from an abandoned copper mine (“Ferrovum myxofaciens” strain P3G) have been elucidated. “F. myxofaciens” is an extremely acidophilic, psychrotolerant obligate autotroph that appears to use only ferrous iron as an electron donor and oxygen as an electron acceptor. It appears to use the Calvin-Benson-Bassham pathway to fix CO₂ and is diazotrophic. It also produces copious amounts of extracellular polymeric materials that cause cells to attach to each other (and to form small streamer-like growth in vitro) and to different solid surfaces. “F. myxofaciens” can catalyze the oxidative dissolution of pyrite and, like many other acidophiles, is tolerant of many (cationic) transition metals. “F. myxofaciens” and related clone sequences form a monophyletic group within the Betaproteobacteria distantly related to classified orders, with genera of the family Nitrosomonadaceae (lithoautotrophic, ammonium-oxidizing neutrophiles) as the closest relatives. On the basis of the phylogenetic and phenotypic differences of “F. myxofaciens” and other Betaproteobacteria, a new family, “Ferrovaceae,” and order, “Ferrovales,” within the class Betaproteobacteria are proposed. “F. myxofaciens” is the first extreme acidophile to be described in the class Betaproteobacteria.     

Temperature Effects on Pea Plants Probed by Simultaneous Measurements of the Kinetics of Prompt Fluorescence,Delayed Fluorescence and Modulated 820 nm Reflection

Simultaneous in vivo measurements of prompt fluorescence (PF), delayed fluorescence (DF) and 820-nm reflection (MR) were made to probe response of pea leaves to 40 s incubation at high temperatures (25–50°C). We interpret our observation to suggest that heat treatment provokes an inhibition of electron donation by the oxygen evolving complex. DF, in a time range from several microseconds to milliseconds, has been thought to reflect recombination, in the dark, between the reduced primary electron acceptor Q_A ^– and the oxidized donor (P680⁺) of photosystem II (PSII). The lower electron transport rate through PSII after 45 and 50°C incubation also changed DF induction. We observed a decrease in the amplitude of the DF curve and a change in its shape and in its decay. Acceleration of P700⁺ and PC⁺ re-reduction was induced by 45°C treatment but after 50°C its reduction was slower, indicating inhibition of photosystem I. We suggest that simultaneous PF, MR and DF might provide useful information on assessing the degree of plant tolerance to different environmental stresses.     

Effects of temperature pretreatment in the dark on photosynthesis of the intact spinach chloroplast

Isolated, intact spinach (Spinacia oleracea L. var. “Long Standing Bloomsdale”) chloroplasts were heated in the dark and the effect of this treatment on photosynthetic activities was determined at 25°C. Dark incubation of the chloroplasts for 10 minutes at 35°C and pH 8.1 resulted in a 50% decline in CO₂ photoassimilation. This decline in photosynthetic performance was dependent upon time, temperature, and medium pH with the optimum effect at acidic pH values. Photosynthetic decline was not observed if MgATP, MgADP, or a mixture of fructose 1,6-bisphosphate, aldolase, and oxaloacetate or ribose 5-phosphate and oxaloacetate was added prior to but not after the temperature pretreatment. A chloroplast preparation reconstituted with thylakoids and stroma from pretreated (35°C, 10 minutes, pH 8.1) intact chloroplasts and supplemented with ferredoxin, ADP, and NADP was photosynthetically competent, indicating that ATP-coupled electron flow and the enzymes comprising the Benson-Calvin cycle remained stable during the dark treatment. In contrast, exposure of isolated thylakoids to 35°C for 10 minutes uncoupled photophosphorylation from NADP and ferricyanide reduction. We propose that the decline of intact chloroplast photosynthesis is the result of a decrease in the content of or a change in the ratios of the adenine nucleotides. Maintenance of an adequate supply of adenine nucleotide is the effect of the externally added MgATP or of chloroplastic respiration of a sugar phosphate.     

Photosynthetic activities in the petunia corolla

Pink Petuniahybrida (cv Hit Parade Rosa) corollas were found to contain photosynthetically active chloroplasts. The corolla chloroplasts were similar to those of green leaves in size and structure. The chlorophyll (Chl) content of Petunia corollas increased during early stages of flower development, reaching a maximum just before anthesis. Chloroplasts isolated from corollas at this stage, carried out photosystem I-dependent electron transport at rates which were two-thirds of those measured in chloroplasts from green leaves, but full chain electron transport at only one-quarter of the rate carried out by chloroplasts from green leaves. Both the light saturated rate and the quantum yield for electron transport were lower in corolla chloroplasts, which also required lower intensities for light saturation. Reduced efficiency of photosystem II photoreactions in the corolla was also indicated by the ratio between variable and constant components of Chl fluorescence, which was lower in corollas compared to green leaves. The induction time of Chl fluorescence was at least three times shorter in corollas compared to green leaves, indicating a smaller number of functional photosystem II centers (per Chl) in the corolla. It is suggested that corolla chloroplasts of Petunia might have a role in flower developmental processes.     

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.     

Theoretical fluorescence induction curves derived from coupled differential equations describing the primary photochemistry of photosystem II by an exciton-radical pair equilibrium

Fluorescence induction curves were calculated from a molecular model for the primary photophysical and photochemical processes of photosystem II that includes reversible exciton trapping by open (PHQ_A) and closed (PHQ^-_A) reaction centers (RCs), charge stabilization as well as quenching by oxidized (P⁺HQ^(-)_A) RCs. For the limiting case of perfectly connected photosynthetic units (“lake model”) and thermal equilibrium between the primary radical pair (P⁺H^-) and the excited singlet state, the primary reactions can be mathematically formulated by a set of coupled ordinary differential equations (ODE). These were numerically solved for weak flashes in a recursive way to simulate experiments with continuous illumination. Using recently published values for the molecular rate constants, this procedure yielded the time dependence of closed RCs as well as of the fluorescence yield (= fluorescence induction curves). The theoretical curves displayed the same sigmoidal shapes as experimental fluorescence induction curves. From the time development of closed RCs and the fluorescence yield, it was possible to check currently assumed proportionalities between the fraction of closed RCs and either (a) the variable fluorescence, (b) the complementary area above the fluorescence induction curve, or (c) the complementary area normalized to the variable fluorescence. By changing selected molecular rate constants, it is shown that, in contrast to current beliefs, none of these correlations obeys simple laws. The time dependence of these quantities is strongly nonexponential. In the presence of substances that quench the excited state, the model predicts straight lines in Stern-Volmer plots. We further conclude that it is impossible to estimate the degree of physical interunit energy transfer from the sigmoidicity of the fluorescence induction curve or from the curvature of the variable fluorescence plotted versus the fraction of closed RCs.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Membranes of Saccharomyces cerevisiae

A crude small particle pellet, obtained from postmitochondrial supernatant fractions of Saccharomyces cerevisiae, contains about half the ergosterol and phospholipid of crude cell homogenates. Most of the phospholipid of this pellet is in a “heavy” fraction which, with the aid of electron microscopy, shows membranous elements in addition to discrete particles. The “heavy” fraction, upon treatment with deoxycholate, can be freed of membranes, or, with ribonuclease treatment, ribosomes can be removed, leaving relatively clean membranes. The “heavy” fraction resembles the microsomes of animal cells, but contains considerably less lipids, including phospholipids, thus suggesting a less well-developed intracellular membrane system.     

Measurement of the fluorescence lifetime of chlorophyll a in vivo

New measurements have been made of fluorescence lifetime (τ) of chlorophyll a in the algae Chlorella pyrenoidosa, Porphyridium cruentum, Anacystis nidulans, and in spinach chloroplast. τ-values of 0.6 and 0.7 nsec were obtained with green plants. Anacystis and Porphyridium gave a τ of 0.5 nsec. The previously described two stage decay of fluorescence in vivo in these organisms could not be confirmed. This observation could have been caused by a second wave of light emission from the exciting hydrogen lamp (not detected in earlier work). The lifetimes found in this study (calculated, as before, by the method of convolution integrals) were close to those found by other observers for “low” excitation intensities; the value first reported from this laboratory (1.0-1.7 nsec) may have corresponded to “high” excitation intensity.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: A Connected Model of the Photosynthetic Unit

The concept of photosynthetic unit (PSU) is reviewed in the light of the authors' results in the fields of fluorescence and luminescence (delayed light). Models of PSU are mainly distinguished by the amount of exciton exchange which is allowed between units. The “separate” model, with its “first-order” character, is not consistent with fluorescence kinetic data. The sigmoidal rise of fluorescence under actinic light is best explained by “nonseparate” models; however, most of these models assume a delocalization of excitons or centers. The “connected” model introduced here is not subject to this criticism. It discloses a new effect (the “îlot” effect): a nonrandom grouping of fluorescent units the consequences of which are discussed. It is noted that a “two-quantum” model for the photochemical reaction gives results very similar to those of the connected model. A relation between luminescence intensity and fluorescence yield is seen as a necessary consequence of the PSU concept. Its meaning is different in separate and nonseparate models. This relation is discussed in connection with the true system II fluorescence emission.     

Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secundatum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts.     

Diurnal Fluctuations in the Content and Functional Properties of the Light Harvesting Chlorophyll a/b Complex in Thylakoid Membranes : Correlation with the Diurnal Rhythm of the mRNA Level

Diurnal fluctuations were observed in the content and some structural and functional properties of the light-harvesting chlorophyll (Chl) a/b pigment-protein complex of photosystem II (LHCII) in young developing wheat (Triticum aestivum) leaves grown under 16 hours light/8 hours dark illumination regime. The fluctuations could be correlated with the diurnal oscillation in the level of mRNA for LHCII. The most pronounced changes occurred in the basal segments of the leaves. They were weaker or hardly discernible in the middle and tip segments. As judged from the diurnal variations of the Chl-a/Chl-b molar ratio, the LHCII content of the thylakoid membranes peaked around 2 pm. This can be accounted for by the cumulative effect of the elevated level of mRNA in the morning and early afternoon. In the basal segment, the extent of the fluctuation in the LHCII content was approximately 25%, as determined from gel electrophoresis (“green gels”). The amplitude of the principal bands of the circular dichroism (CD) spectra of isolated chloroplasts paralleled the changes in the LHCII content. Our circular dichroism data suggest that the newly synthesized LHCII complexes are incorporated into the existing helically organized macrodomains of the pigment-protein complexes or themselves form such macrodomains in the thylakoid membranes. Chl-a fluorescence induction kinetics also showed diurnal variations especially in the basal segments of the leaves. This most likely indicates fluctuations in the ability of membranes to undergo “state transitions.” These observations suggest a physiological role of diurnal rhythm of mRNA for LHCII in young developing leaves.     

Effects of Manganese Deficiency and Added Cerium on Photochemical Efficiency of Maize Chloroplasts

The mechanism of the fact that manganese deprivation and cerium addition affect the photochemical efficiency of plants is unclear. In this study, we investigated the improvement by cerium of the damage of the photochemical function of maize chloroplasts under manganese-deprived stress. Chlorophyll fluorescence induction measurements showed that the ratio of variable to maximum fluorescence (Fv/Fm) underwent great decreases under manganese deficiency, which was attributed to the reduction of intrinsic quantum efficiency of the photosystem II units. The electron flow between the two photosystems, activities of Mg²⁺–ATPase and Ca²⁺–ATPase, and rate of photophosphorylation on the thylakoid membrane of maize chloroplasts were reduced significantly by exposure to manganese deprivation. Furthermore, the inhibition of cyclic photophosphorylation was more severe than non-cyclic photophosphorylation under manganese deficiency. However, added cerium could relieve the inhibition of the photochemical reaction caused by manganese deprivation in maize chloroplasts. It implied that manganese deprivation could disturb photochemical reaction of chloroplasts strongly, which could be improved by cerium addition.     

α-Proteobacterial Symbionts of Marine Bryozoans in the Genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of “W. subtorquata” and “W. arcuata” from several locations along the California coast are actually closely related α-Proteobacteria, not mollicutes. We propose the names “Candidatus Endowatersipora palomitas” and “Candidatus Endowatersipora rubus” for the symbionts of “W. subtorquata” and “W. arcuata,” respectively.     

The Pine Bark Adelgid,Pineus strobi,Contains Two Novel Bacteriocyte-Associated Gammaproteobacterial Symbionts

Bacterial endosymbionts of the pine bark adelgid, Pineus strobi (Insecta: Hemiptera: Adelgidae), were investigated using transmission electron microscopy, 16S and 23S rRNA-based phylogeny, and fluorescence in situ hybridization. Two morphologically different symbionts affiliated with the Gammaproteobacteria were present in distinct bacteriocytes. One of them (“Candidatus Annandia pinicola”) is most closely related to an endosymbiont of Adelges tsugae, suggesting that they originate from a lineage already present in ancient adelgids before the hosts diversified into the two major clades, Adelges and Pineus. The other P. strobi symbiont (“Candidatus Hartigia pinicola”) represents a novel symbiont lineage in members of the Adelgidae. Our findings lend further support for a complex evolutionary history of the association of adelgids with a phylogenetically diverse set of bacterial symbionts.