Acceleration of dark reversion of phytochrome in vitro by calcium and magnesium

Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples.     

A detailed analysis of phytochrome decay and dark reversion in mustard cotyledons

Summary During the first 10 min after a saturating dose of red light, 72 h dark-grown mustard cotyledons show no phytochrome decay. Within the same time interval there exists a transient form of P _fr (=P _fr ^T ) which is no longer photoconvertible at 0°C, but is at 25°C. This P _fr ^T converts in the dark to P _fr and P _r . These dark reversions take about 10 min. After a lag phase of 10 min the P _fr decay can be described by a single, first order kinetic curve. The time courses of these reactions are functions of the time of etiolation.Research supported by DAAD and by Deutsche Forschungsgemeinschaft (SFB 46).     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

The dark reactions of rye phytochrome in vivo and in vitro

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo. The effects of various inhibitors of an in vitro phytochrome-degrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 molecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.     

Phototransformation of phytochrome in the dark

Enzymatically generated triplet acetone transfers its energy to the ground state phytochrome and promotes to some extent, in the dark, the conversion of P_r into P_fr and of P_fr into P_r. This is the first report of inverse dark reversion “in vitro”.     

In vitro photomodulation of a peroxidase activity through membrane-bound phytochrome

Membrane vesicles are prepared from hypocotyl hooks of dark-grown Cucurbita pepo. Phytochrome is bound to these membranes by a 4-min pretreatment with red light during the extraction procedure. The peroxidase activity associated with membranous vesicles is then measured after various light treatments in vitro. It appears that red light (660 nm) lowers the activity in a membrane suspension containing phytochrome, but has practically no effect if phytochrome was not previously bound by a red pretreatment. Far red light reverses this effect, with a maximum efficiency about 720 nm. This phytochrome-mediated photocontrol of an enzymatic activity suggests that the binding of the pigment to membranes has a physiological significance.     

Dependence of phytochrome dark reactions on the initial photostationary state

Summary Under conditions of continuous irradiation, the P _jr destruction rate constants (k _d ) of phytochrome in hooks and cotyledons of squash (Cucurbita pepo L.) seedlings do not depend on the photostationary state and are the same in both organs. On the other hand, the rate constants of the dark reversion and the first destruction step, plotted as a function of ⁰ , show optimum curves with maxima between ⁰ and 0.5. Similar results were obtained for dark reactions of mustard (Sinapis alba L.)-hook phytochrome in vivo. This indicates a cooperative behaviour of these phytochrome dark reactions.Abbreviations P _r red-absorbing form of phytochrome - P _fr far-red-absorbing form of phytochrome - [P _tot] [P _r ]+[P _fr ] - [P _tot] ([P _fr ]/[P _tot]), photostationary state - ⁰ at t=0, immediately after saturating irradiation     

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.     

白念珠菌对特比萘芬耐药性的体外梯级诱导和回复研究

目的以浓度梯级倍增的特比萘芬在体外诱导白念珠菌标准株获得耐药子代菌株,并观察其耐药稳定性,从细胞水平研究白念珠菌对特比萘芬耐药前后生物学特性的变化,为进一步采用基因芯片在基因表达水平上研究特比萘芬对白念珠菌的药理作用及其诱导耐药机制提供理想的实验模型。方法将白念珠菌ATCC90028株在特比萘芬浓度逐渐梯级倍增的YPD液体培养基中分别转种传代,直到最后转种至含1024μg／ml特比萘芬的YPD液体培养基中培养,分别测定诱导后形成的各子代菌株的MIC值;选用以1024μg／ml特比萘芬诱导形成的耐药菌株,在不含特比萘芬的YPD液体培养基中连续传代10次后,测定其MIC值,观察其耐药表型的稳定性;并分别用肉眼、光镜和电镜观察白念珠菌耐药性产生前后的形态学特征。结果特比萘芬MIC值为8μg／ml的白念珠菌母本菌株（白念珠菌ATCC90028）成功地被诱导成特比萘芬MIC值为≥512μg/ml的子代菌株,进一步的耐药稳定性实验说明诱导后形成的子代菌株的表型是相对稳定的,诱导后的子代耐药菌株与其母本相比,生长繁殖速度减慢,细胞形态不规则,部分细胞胞膜不完整。结论通过在药物浓度梯级倍增的培养基中连续传代培养的方法可成功建立相同基因型的对特比萘芬敏感的白念珠菌母本和对特比萘芬耐药的子代模型,为获取有亲本的耐药白念珠菌菌株提供了一个有效的实验方法,是在基因水平研究白念珠菌对特比萘芬耐药机制的理想实验模型。     

The loss of phytochrome photoreversibility in vitro

Killer, a substance extracted from stem tissue of etiolated pea seedlings (Pisum sativum L. v. Alaska), interacts specifically with the far-red-absorbing form of phytochrome (Pfr) in vitro in a temperature-independent, rapid, stoichiometric fashion to cause a loss of phytochrome photoreversibility. The chromatographic, solubility, and spectral properties of partially purified fractions indicate that Killer is a cyclic, unsaturated molecule containing ionizible hydroxyl groups; its molecular weight is unknown, although probably low. Possible mechanisms by which the Killer-phytochrome interaction results in the loss of photoreversibility are discussed.I=Fox, 1975     

In vivo measurement of phytochrome in tomato fruit

Presence of phytochrome in two kinds of tomatoes (Lycopersicon esculentum Mill.), the yellow lutescent strain and cherry tomatoes (L. esculentum Mill. var. cerasiformecv. Red Cherry), was established by measuring the absorption difference spectra of the whole fruit after irradiation with red and with far red light. Phytochrome content was determined in yellow lutescent tomatoes and decreased gradually during the ripening period.     

Binding of phytochrome to plasma membranes in vitro

Nicotiana tabacum (cv. Xanthi), N. plumbaginifolia Viviani, N. sylvestris Speg. and Comes and Petunia axillaris × ( P. axillaris × P. hybrida ) (cv. Mitchell) mesophyll protoplast-derived cells were able to grow at low densities in chemically-defined media. Protoplasts of different origins (mesophyll, epidermis, pith, suspension culture) also gave rise to protoplast-derived cells that were able to grow at low densities. When auxin requirements at low densities were compared for different sources of auxins, IAA was found to be efficient in the same range of concentrations as NAA. This was unexpected since tobacco mesophyll protoplasts cannot be induced to divide when plated at high density in the presence of IAA. Optimal 2,4-D concentrations for low density growth were higher and clearly pH-dependent. On the contrary, picloram induced low density growth over a wide range of concentrations suggesting a distinct mechanism of action. These results confirm and extend previous observation on the tobacco mesophyll protoplast and show that the low-density growth technique has a potential use for the study of the action of phytohormones.     

Photochemistry of 124 kilodalton Avena phytochrome in vitro

The photochemical properties of purified 124 kilodalton (kD) Avena cv Garry phytochrome are examined and compared with those of the proteolytically degraded 118/114 kD species. The proportion of the chromoprotein in the far red absorbing form, Pfr, following saturating red irradiation is 0.86 for 124 kD phytochrome, substantially higher than the values of 0.79 determined here and 0.75 reported in the literature for 118/114 kD preparations. The ratio of the quantum yields for Pr to Pfr phototransformation and for Pfr to Pr phototransformation (r/fr) is 1.76 for the 124 kD molecule and 0.98 for the 118/114 kD species. Based on extinction coefficients determined using the Lowry assay as a measure of protein weight, the individual phototransformation quantum yields for 124 kD phytochrome are 0.17 for Pr → Pfr (r) and 0.10 for Pfr → Pr (fr). Comparison of these quantum yields with those of the 118/114 kD species (where r = fr = ~0.11) indicates that proteolytic degradation of the 124 kD molecule to the 118/114 kD species significantly affects only r. Therefore, the lower proportion of Pfr at photoequilibrium observed for 118/114 kD preparations is explained mainly in terms of a reduced efficiency of Pr → Pfr phototransformation. The absolute Pfr absorbance spectrum for 124 kD phytochrome obtained by correcting the measured spectrum for residual Pr exhibits a maximum at 730 nm and differs from previous absolute Pfr spectra for both `120' kD and 60 kD phytochrome in that it lacks a shoulder in the red region of the spectrum.