Excited-state dynamics of protochlorophyllide revealed by subpicosecond infrared spectroscopy

To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ₁ = 3.6 ± 0.2, τ₂ = 38 ± 2, and τ₃ = 215 ± 8 ps. As indicated by the C13′=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ₂ describes the formation of a state with a strong change in electronic structure, and τ₃ represents the partial recovery of the PChlide electronic ground state. Furthermore, τ₁ corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.     

Far-red induced changes of the protochlorophyllide components in wheat leaves

Biosynthesis of chlorophyll is partly controlled by the phytochrome system. In order to study the effects of an activated phytochrome system on the protochlorophyllide (PChlide) biosynthesis without accompanying phototransformation to chlorophyll, wheat seedlings (Triticum aestivum L. cv. Starke II Weibull) were irradiated with long wavelength far-red light of low intensity. Absorption spectra were measured in vivo after different times in the far-red light or in darkness. The relationship between the different PChlide forms, the absorbance ratio _{650nm636 nm} changed with age in darkness, and the change was more pronounced when the leaves were grown in far-red light. Absorption spectra of dark-grown leaves always showed a maximum in the red region at 650 nm. For leaves grown in far-red light the absorption at 636 nm was high, with a maximum at the 5 day stage where it exceeded the absorption at 650 nm. At the same time there was a maximum in the total amount of PChlide accumulated in the leaves, about 30% more than in leaves grown in darkness. But the amount of the directly phototransformable PChlide, mainly PChlide_650–657, was not increased. The amount of PChlide_628–632, or more probably the amount of (PChlide_628–632, + PChlide _636–657) was thus higher in young wheat leaves grown in far-red light than in those grown in darkness. After the 5 day stage the absorption at 636 nm relative to 650 nm decreased with age, and at the 8 day stage the spectra were almost the same in both types of leaves. Low temperature fluorescence spectra of the leaves also showed a change in the ratio between the different PChlide forms. The height of the fluorescence peak at 632 nm relative to the peak at 657 nm was higher in leaves grown in far-red light than in dark-grown leaves. – After exposure of the leaves to a light flash, the half time for the Shibata shift was measured. It increased with age both for leaves grown in darkness and in far-red light; but in older leaves grown in far-red light (7–8 days) the half time was slightly longer than in dark-grown leaves. – The chlorophyll accumulation in white light as well as the leaf unrolling were faster for leaves pre-irradiated with far-red light. The total length of the seedlings was equal or somewhat shorter in far-red light, but the length of the coleoptile was markedly reduced from 8.1 ± 0.1 cm for dark-grown seedlings to 5.2 ± 0.1 cm for seedlings grown in far-red light.     

Energetics of growth of Microbacterium thermosphactum at low temperatures

Microbacterium thermosphactum was grown at 5°C and 9°C in glucose-limited continuous cultures. The end products of glucose metabolism were L-lactate and ethanol, and these compounds accounted for 86–92% of the glucose utilized. With input glucose concentrations less than 3 mM Y _glu ^Max was found to be 40–43, Y _ATP ^Max 20–21 and m _s 0.1–0.2. These values are almost identical to those found previously for cultures at 25°C and show that this psychrotroph grows with a very high energetic efficiency over a wide range of temperatures. With a higher (but still limiting) input glucose concentration of 5.6 mM at 9°C, cellular efficiency declined as there was a marked reduction in Y _glu. This decrease was accounted for in mathematical terms by an increase in m _s to 0.7, whilst Y _glu ^Max and Y _ATP ^Max remained high at 38 and 19 respectively.     

Effects of glucose-supplemented diets on food intake, nymphal development, and fecundity of glucose-averse, non-glucose-averse, and heterozygous strains of the German cockroach,Blattella germanica

Life history parameters were determined for glucose-averse (glu/glu), wild-type (glu+/glu+) and heterozygous (glu/glu+) genotypes of Blattella germanica (L.) (Blattodea: Blattellidae) fed diets supplemented with glucose. Glu/glu nymphs consumed less glucose-supplemented diet, gained less weight, developed slower and had a lower rate of survival than glu/glu nymphs fed the same diet without added glucose, or glu+/glu+ and glu/glu+ fed either diet. Prior to formation of the first oötheca, female glu/glu consumed less glucose-supplemented diet per day than glu+/glu+ and glu/glu+, which presumably delayed egg case production. Oötheca-bearing glu/glu and glu/glu+ females consumed less glucose-supplemented diet than glu+/glu+ females. Despite a difference in female total diet intake, there was no effect of diet or genotype on fecundity. However, the intrinsic rate of increase (r) for glulglu on unsupplemented diet was less than that of glu+/glu+ and glu/glu+, suggesting that individuals with both glu alleles may be at a selective disadvantage in environments lacking diets containing glucose plus a toxicant.     

Control of chlorophyll synthesis by phytochrome

Summary The rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (P_fr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that P_fr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by P_fr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55–58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of P_fr.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Chl(ide) sum of Chl and Chlide - PChl protochlorophyll - PChlide protochlorophyllide - PChl(ide) sum of PChl and PChlide - P_fr far-red absorbing form of the phytochrome system     

Pheophytinization kinetics of chlorophyll c under weakly acidic conditions: Effects of acrylic acid residue at the 17-position

Pheophytinization of chlorophyll (Chl) c₁, which was isolated from the diatom Chaetoceros gracilis, was kinetically analyzed under weakly acidic conditions, and was compared with that of protochlorophyllide (PChlide) a and chlorophyllide (Chlide) a. Chl c₁ possessing a trans-acrylic acid residue at the 17-position exhibited slower pheophytinization kinetics than PChlide a and Chlide a, both of which possessed a propionic acid residue at the same position. The difference in pheophytinization properties between Chl c₁ and (P)Chlide a was ascribable to the electronegativity of the 17-substituent in Chl c₁ larger than that of (P)Chlide a due to the C17¹–C17² double bond with the conjugated 17²-carboxy group in Chl c₁. Demetalation kinetics of PChlide a was slower than that of Chlide a, which originated from the effect of the π-macrocyclic structures.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

楚科奇海及其海台区粒度分级叶绿素a与初级生产力

2003年夏季中国第二次北极科学考察期间,在楚科奇海及其海台区进行了叶绿素a浓度与初级生产力的现场观测。结果表明,观测海区叶绿素a浓度范围为0.009～30.390μg/dm3。表层浓度为0.050～4.644μg/dm3,平均值为(0.875±0.981)μg/dm3;陆架区次表层和底层的浓度高于表层,海台区深层水的浓度较低,200m层的浓度为(0.015±0.007)μg/dm3。水柱平均叶绿素a浓度区域性特征明显,陆架区高于海台区。R断面进行3趟重复观测,平均叶绿素a浓度分别为(2.564±1.496)μg/dm3,(1.329±0.882)μg/dm3和(0.965±0.623)μg/dm3,浓度呈下降趋势。观测站潜在初级生产力为0.263～4.186mgC/(m.3h),陆架区平均潜在初级生产力((2.305±1.493)mgC/(m.3h))比海台区((0.527±0.374)mgC/(m.3h))高近4倍。平均同化数为(1.22±1.14)mgC/(mgChla.h)。观测区细胞粒径>20μm的小型浮游生物对总叶绿素a浓度和初级生产力的贡献率分别为63.13%和65.16%,细胞粒径2.0～20μm的微型浮游生物和细胞粒径<2.0μm的微微型浮游生物对总叶绿素a和初级生产力的贡献率相差甚小,其对总叶绿素a浓度的贡献率分别为19.18%和17.69%,对总初级生产力的贡献率分别为20.11%和14.73%。     

Filamentous green algae inhibit phytoplankton with enhanced effects when lakes get warmer

1. Filamentous green algae (FGA) may represent an alternative state in high‐nutrient shallow temperate lakes. Furthermore, a clear water state is sometimes associated with the dominance of FGA; however, the mechanisms involved remain uncertain. 2. We hypothesised that FGA may promote a clear water state by directly suppressing phytoplankton growth, mostly via the release of allelochemicals, and that this interaction may be affected by temperature. 3. We examined the relationships between FGA, phytoplanktonic chlorophyll a concentrations and zooplankton in a series of mesocosms (2.8 m³) mimicking enriched shallow ponds now and in a future warmer climate (0 and c. 5 °C above ambient temperatures). We then tested the potential allelopathic effects of FGA (Cladophora sp. and Spirogyra sp.) on phytoplankton using several short‐term microcosms and laboratory experiments. 4. Mesocosms with FGA evidenced lower phytoplanktonic chlorophyll a concentrations than those without. Zooplankton and zooplankton : phytoplankton biomass ratios did not differ between mesocosms with and without FGA, suggesting that grazing was not responsible for the negative effects on phytoplanktonic biomass (chlorophyll a). 5. Our field microcosm experiments demonstrated that FGA strongly suppressed the growth of natural phytoplankton at non‐limiting nutrient conditions and regardless of phytoplankton initial concentrations or micronutrients addition. Furthermore, we found that the negative effect of FGA on phytoplankton growth increased up to 49% under high incubation temperatures. The experiment performed using FGA filtrates confirmed that the inhibitory effect of FGA on phytoplankton may be attributed to allelochemicals. 6. Our results suggest that FGA control of phytoplankton growth may be an important mechanism for stabilising clear water in shallow temperate lakes dominated by FGA and that FGA may play a larger role when lakes get warmer.     

HIGH-RESOLUTION AIRBORNE REMOTE SENSING OF BLOOM-FORMING PHYTOPLANKTON1

Remote sensing of highly turbid finfish aquaculture impoundments using the Calibrated Airborne Multispectral Scanner (CAMS) mounted on a Lear jet flown at 900 m was conducted in central Mississippi on 16 May 1990. Concurrent in situ data consisted of phytoplankton pigment concentrations and standing crop, water color, turbidity, and surface-water temperature. Surface and near-surface assemblages of cyanophytes and chlorophytes varied dramatically among impoundments; total chlorophyll concentrations and standing crop values ranged from 8 to 483 mg·m^?3 and 8.0 × 10² to 2.2 × 10⁶ cells-mL^?1, respectively. Regression models fit to CAMS data provided reliable estimates for and produced accurate digital cartographs of total chlorophyll and carotenoid concentrations, phytoplankton standing crop, and turbidity. Although a model to effectively estimate in situ c-phycocyanin concentrations was not identified, the lack of a suitable model may have resulted from variability of pigment extraction during quantification rather than failure of remotely sensed imagery to detect c-phycocyanin. Models derived from imagery of impoundments directly beneath the aircraft sufficiently described in situ parameters in imagery of adjacent series of impoundments not directly below the aircraft. High-resolution airborne remote sensing provides a means for monitoring local phytoplankton dynamics in temporal and spatial scales analogous to biotic and abiotic processes affecting such dynamics and necessary for applications to ecological research and fisheries or aquacultural management.     

外源5-氨基乙酰丙酸对NaCl胁迫下酸枣光合特性的影响

以2年生酸枣幼苗为试验材料,探讨不同浓度NaCl(0、4、8、12g·kg-1)胁迫下喷施5-氨基乙酰丙酸(ALA,75、150mg·L~(-1))对酸枣光合特性的影响。结果显示:(1)不同浓度NaCl胁迫下,外源ALA对酸枣叶片的净光合速率(Pn)、胞间二氧化碳浓度(Ci)、气孔导度(Gs)、蒸腾速率(Tr)及叶绿素含量等具有明显促进作用。(2)在不同浓度NaCl胁迫下,喷施75mg·L~(-1)的ALA仅在NaCl浓度为4和8g·kg-1处理下对酸枣Tr值具有显著提高作用,而喷施150mg·L~(-1) ALA在各NaCl浓度胁迫下对其Pn、Ci和Tr均具有显著促进作用。(3)在重度NaCl胁迫(12g·kg-1)下,喷施150mg·L~(-1) ALA对酸枣叶片叶绿素含量具有显著提高作用,而喷施75mg·L~(-1)浓度ALA却无明显提高。研究表明,在NaCl胁迫条件下,外源ALA能有效改善酸枣叶片光合气体交换参数,提高叶绿素含量,从而缓解NaCl胁迫的伤害,提高其光合能力,并以喷施150mg·L~(-1) ALA的缓解效果更好。     

Aspects toxiques du cuivre sur la biomasse et la productivité du phytoplancton de la rivière du Saguenay,Québec

In 1979 and 1980, batch culture experiments were conducted to observe the inhibitory effect of copper ion (concentrations of 10, 50, 100, 200 and 400 µg Cu · l^–1) on the standing crops and photosynthesis of phytoplankton of the Saguenay River (for 124 hours) and in Chlorella vulgaris (for 8 days). These algal assays were carried out using the surface water of the Saguenay River. In natural populatoins of phytoplankton, it was found that photosynthesis was more sensitive than growth: at the lowest concentrations, such as 10 µg Cu · 1^–1, copper seemed to increase the chlorophyll concentrations whereas the rates of primary production show a decrease of 60% with respect to the control. At higher concentrations of copper, the effect is weak in chlorophyll concentrations and more pronounced in the rates of primary production (decrease of 86 to 90%). The pennate diatoms are dominant (in all the samples) and these organisms are known as relatively resistant to copper. In Chlorella vulgaris, it was observed that with 100 µg Cu · 1^–1, chlorophyll concentrations and rates of photosynthesis respectively decrease by 63 and 99% with respect to the control. At higher concentrations of copper, a maximum decrease of 70% and 99% respectively for chlorophyll concentrations and rates of primaryproduction are observed.

     

Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Euglena gracilis (Euglenophyceae) produces abscisic acid and cytokinins and responds to their exogenous application singly and in combination with other growth regulators

The main objective of this study was to determine the optimal concentrations of a wide spectrum of exogenous phytohormones for effective stimulation of cell division and production of maximum cell yield in Euglena gracilis Klebs cultured in vitro. Results indicate that two hormones combined exert more effective growth stimulation than a single hormone or three, four or five different hormones combined. Specifically, trans-zeatin at 10^?7 M combined with abscisic acid at 10^?9 M produced optimal conditions for growth, yielding the maximum cell concentration. High concentrations of exogenous phytohormones were toxic to Euglena. The addition of trans-zeatin, N6-isopentenyladenine, and benzylaminopurine to Euglena cultures resulted in dense, dark green chloroplasts, suggesting that exogenous phytohormones increased the production of chlorophyll. Given the response to exogenous growth regulators, the study identified and quantified the types of endogenous cytokinins (CKs) and abscisic acid (ABA) synthesized in vitro by Euglena gracilis. HPLC-(ESI) MS/MS analysis revealed that the algal cells produced and released into the medium a mixture of CKs and ABA. The main CKs identified in the cell pellets and supernatant samples were from a t-RNA degradation pathway and included: cis-zeatin (cZ) derivatives cZR, cZNT, MeSZ and MeSZR, and to a lesser extent, the free base N6-isopentenyladenine (iP) and its derivatives iPR, iPNT, MeSiP and MeSiPA. A positive response to ABA, and the relatively high levels detected in E. gracilis, suggest that this hormone is important for alleviating stress conditions of in vitro culture that might otherwise restrict cell division.     

Immune responses of mice against poly(glu lys ala) terpolymers and linkage with H-2

The immune responses of inbred mice to the terpolymers poly(glu⁴⁸-Iys³² ala²⁰) GLA²⁰, poly(glu³⁶lys²⁴ala⁴⁰) GLA⁴⁰, and poly(glu²⁴lys¹⁶ala⁶⁰) GLA⁶⁰ were studied. Antibody levels were measured with the homologous, as well as with the crossreacting polymers (glu⁶⁰ala⁴⁰) GA and (glu⁶⁰lys⁴⁰) GL. It was determined that the terpolymers consist of many determinants of varying immunogenic strengths which account for the dose dependency requirements for responsiveness as follows: mice ofH-2 haplotypesa, b, d, k, ands respond to ten and 100g GLA²⁰ and GLA⁴⁰ and to one, ten, and 100 g GLA⁶⁰; mice ofH-2 haplotypesp, q, andr do not respond well to any concentration of GLA²⁰ but respond well to 100 g GLA⁴⁰ and teng GLA⁶⁰. That the congenic mice C3H.NB (H-2 ^p) and B10.R111 (H-2 ^r), having responder backgrounds of C3H (H-2 ^k) and C57BL/10 (H-2 ^b) mice, respectively, do not respond would suggest strongly that there is linkage of responsiveness toH-2 in the above strains. In addition, the responsiveness of AQR mice to GLA⁶⁰ would map theIr gene(s) to the right of theK region, and most likely in theI region. The antibody against GLA²⁰ was directed against GL. Responses of miceof H-2 haplotypesp, q, andr against GLA⁴⁰ and GLA⁶⁰ were directed predominantly against unique GLA determinants that were neither GA nor GL. Mice of the other respondingH-2 haplotypes (a, b, d, k, ands) produced antibody against these unique GLA specificities, as well as against GL and/or GA determinants. The importance of measuring responses with the homologous polymer is therefore demonstrated. It was postulated that the recognition of GLA²⁰ at the T-cell level is via GLA determinants having a limited amount of alanine, which are different from those helical GLA determinants recognized in the polymers GLA⁴⁰ and GLA⁶⁰.     

Adaptation of epiphytic bryophytes in the understorey attributing to the correlations and trade-offs between functional traits

This study explores adaptive strategies of epiphytic bryophytes in the understorey by investigating the photosynthetic characteristics, pigment concentrations and nutrient stoichiometry, as well as other functional traits of three trunk-dwelling bryophytes in a subtropical montane cloud forest in SW China. The results showed that their light-saturated net photosynthetic rate (A_nmax?L), light saturation point (I_sat), light compensation point (I_c) and dark respiration rate (R_d) were ca 0.55, 106.72, 4.17 and 0.25?μmol?m^?2?s^?1, respectively. Furthermore, the samples demonstrated photosynthetic down-regulation under high irradiance. These photosynthetic characteristics can be explained by higher total chlorophyll concentrations, specific leaf area, chlorophyll per unit leaf N (Chl/N), lower ratio of chlorophyll a to chlorophyll b (Chl a/b) and photosynthetic nitrogen-use efficiency. We suggest that the bryophytes adapted to the shaded understorey microhabitats through a series of correlations and trade-offs between functional traits.     

Effect of Ni2+ on Early Stages of Chlorophyll Biosynthesis and Pheophytinization in Euglena gracilis

The effect of Ni²⁺ on the early stages of chlorophyll biosynthesis and pheophytinization in Euglena gracilis cells was studied. Incubation of the cells with 10^–4 M Ni²⁺ for 7 days resulted in a higher chlorophyll content, enhanced production of 5-aminolevulinic acid (ALA), and in increased activity of 5-aminolevuluinic acid dehydratase (EC 4.2.1.24, ALAD), as compared to the control cells incubated without Ni²⁺. At a higher concentration (10^–3 M), Ni²⁺ markedly inhibited chlorophyll accumulation and ALAD activity, as compared to the control cells. At this concentration, Ni²⁺ also inhibited heme biosynthesis and strongly stimulated ALA production. It seems likely that, by affecting heme synthesis, Ni²⁺ increases the activity of the ALA production system. However, the suppression of subsequent stages of ALA conversion to chlorophyll, in particular ALAD inhibition, ultimately resulted in almost complete inhibition of chlorophyll biosynthesis. In addition to cessation of de novo chlorophyll synthesis in the presence of Ni²⁺ (10^–3 M) in Euglena cells, the existing chlorophyll was converted into pheophytin and almost completely degraded. We suppose that the Ni²⁺-induced pheophytinization is caused by an acidic shift of intracellular pH related to an impairment of cell membrane permeability by Ni²⁺ cations.     

Promotion and inhibition by ethylene of chlorophyll degradation in orange fruits

The effect of ethylene concentration on chlorophyll destruction in orange (Citrus sinensis cV. Washington Navel) fruits was examined at 15 °C and 25 °C. The reflectance of the fruits at 680 nm was measured, and the results converted to chlorophyll concentrations through an empirically derived formula based on the Kubelka-Munk equation. In all ethylene treatments chlorophyll destruction was faster at 25 °C than at 15 °C and all ethylene concentrations tested increased the rate of loss at 25 °C. At 15 °C the highest rate of chlorophyll destruction was observed at 1 μl litre^-1 ethylene, while chlorophyll loss at 1250 μl litre^-1 was slower than in untreated fruits.     

Regulation of the Na+-coupled glutamate transporter EAAT3 by PIKfyve

The Na⁺,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I_glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I_glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. The increased I_glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I_glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.