Differences in Photoresponse and Phytochrome Spectrophotometry Between Etiolated and De-etiolated Pea Stem Tissue

Morphologically similar pea plants having a 4-fold difference in spectrophoto-metrically detectable phytochrome can be produced by pretreatment of etiolated plants with red light (R) or with red and far-red light combined (RF). A search for response differences which could be ascribed to differences in phytochrome content has resulted only in the establishment of differences due to de-etiolation. Segments of etiolated plants differ from those of plants de-etiolated by R and RF pretreatments in 2 ways. Segments from etiolated plants appear to respond rapidly to the far-red absorbing form of phytochrome (P_FR), while segments from de-etiolated plants do not respond rapidly to P_FR. This statement is based upon 2 observations: (i) the red light induced growth inhibition in segments from etiolated plants rapidly escapes reversibility by far-red light, while with segments from R or RF pretreated plants, the red light effect is fully reversed by subsequent far-red light for up to 2 hr; and (ii) segments from etiolated plants were inhibited to a greater degree than were segments from RF pretreated plants when various photostationary state levels of P_FR were maintained for 30 or 90 min and then removed by photoconversion to P_R. The in vivo nonphotochemical transformation curves of the phytochrome of etiolated and RF pretreated plants appear to differ in 2 related respects: (i) the amount of phytochrome destroyed in de-etiolated tissue is greater than that in etiolated tissue, perhaps as a result of the fact that (ii) the rate and extent of apparent reversion of P_FR to P_R in etiolated tissue is about twice that in de-etiolated tissue.     

Phytochrome in cultured wild carrot tissue. I. Synthesis

Stable levels of P_FR occur in light-grown suspension cultures of wild carrot. Upon darkening such tissue, P_R accumulates until the high P_R level characteristics of dark-grown tissue is reached. Data are compatible with the idea of an equilibrium between processes of synthesis and destruction in tissue grown in the light. Reappearance of phytochrome also follows light-induced pigment destruction in dark-grown tissue. The rate of P_R appearance is similar in both cases. The rate of disappearance of P_FR, however, is slower in light-grown tissue than in dark-grown tissue.     

Some Properties of Phytochrome Isolated From Dark-grown Oat Seedlings (Avena sativa L.)

Phytochrome was partially purified from etiolated seedlings of Avena sativa L. Several properties of the red-absorbing (P_R) and far-red absorbing (P_FR) forms of the pigment were compared. The 2 forms could not be shown to differ with respect to their sedimentation velocity in sucrose density gradients, elution volume from Sephadex G-200 columns, binding properties on calcium phosphate, or electrophoretic mobility. P_FR, however, was more labile than P_R during precipitation with 50% ammonium sulfate. Sephadex G-200 elution diagrams obtained with fresh phytochrome preparations revealed 2 components of different molecular weights, 1 roughly 180,000, and 1 roughly 80,000. Native phytochrome had an absorption spectrum in vivo showing an absorption maximum for P_R of 667 nm. Both the large and small forms of phytochrome mentioned above can be maintained with an absorption maximum for P_R of 667 nm. However, allowing them to remain for several hours as P_FR, even at 4°, shifted this peak to 660 nm. The protein conformational change during phytochrome transformation may be quite small, though the various comparative techniques used do not strictly rule out a fairly large one. The need for maintaining the pigment as P_R during all steps of purification, but particularly during ammonium sulfate precipitation is underscored.     

Genetic Regulation of Development in Sorghum bicolor: VI. The ma(3) Allele Results in Abnormal Phytochrome Physiology

Physiological processes controlled by phytochrome were examined in three near-isogenic genotypes of Sorghum bicolor, differing at the allele of the third maturity gene locus. Seedlings of 58M (ma₃^R ma₃^R) did not show phytochrome control of anthocyanin synthesis. In contrast, seedlings of 90M (ma₃ma₃) and 100M (Ma₃Ma₃) demonstrated reduced anthocyanin synthesis after treatment with far red and reversal of the far red effect by red. De-etiolation of 48-hour-old 90M and 100M dark-grown seedlings occurred with 48 hours of continuous red. Dark-grown 58M seedlings did not de-etiolate with continuous red treatment. Treatment of seedlings with gibberellic acid or tetcyclacis, a gibberellin synthesis inhibitor, did not alter anthocyanin synthesis. Levels of chlorophyll and anthocyanin were lower in light-grown 58M seedlings than in 90M and 100M. Etiolated seedlings of all three genotypes have similar amounts of photoreversible phytochrome. Crude protein extracts from etiolated seedlings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phytochrome was visualized with Pea-25, a monoclonal antibody directed to phytochrome from etiolated peas. The samples from all three genotypes contained approximately equivalent amounts of a prominent, immunostaining band at 126 kD. However, the sample from 58M did not show a fainter, secondary band at 123 kD that was present in 90M and 100M. The identity and importance of this secondary band at 123 kD is unknown. We propose that 58M is a phytochrome-related mutant that contains normal amounts of photoreversible phytochrome and normal phytochrome protein when grown in the dark.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Long-lived Intermediates in Phytochrome Transformation II: In Vitro and In Vivo Studies

Conditions of illumination which cause phytochrome to cycle rapidly from P_R to P_FR and back lead to the accumulation in vivo of detectable amounts of long-lived intermediates on the P_R to P_FR pathway in oat coleoptile tissue. They appear to decay independently and in parallel to P_FR. Their behavior under different intensities of illumination and exposure time suggests that they are homologous with 2 similar intermediates previously observed in vitro. Available evidence favoring this suggestion is discussed. Equivalent illumination apparently causes far higher steady state levels of absorption by intermediates in vivo than in vitro, suggestion that native phytochrome is in a different physical state in the cell than it is in solution. A difference spectrum for the intermediates in vitro between 365 and 580 nm is presented. It has a maximum at 380 nm, a minimum at 418 nm, and crossover points at 398 and 485 nm. Glycerol in the phytochrome sample enhances the signal without otherwise changing the spectrum in any way. The difference spectrum represents the difference in absorption between the combined intermediates and P_FR.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

Aspects of phytochrome decay in etiolated seedlings under continuous Illumination

Summary The rate of total phytochrome decay in the dicotyledons Amaranthus caudatus, Mirabilis jalapa and Pisum sativum under continuous illumination with red, incandescent, and blue light depends on the P_FR/P_total maintained by each source. Amaranthus is an exception to this in that there is a deviation from firstorder decay kinetics under continuous illumination with incancdescent light. This deviation is probably not related to the chlorophyll present in the Amaranthus sample since chlorophyll-rich Pisum buds have the same phytochrome decay rate as epicotyl tissue under continuous incandescent light. Reports of a prolonged lag phase before the onset of first-order decay kinetics of phytochrome in Pisum have not been confirmed and the small lag phase observed in the present work can be accounted for by the time required to attain the P_FR/P_total ratio characteristic of blue light in a carotenoid rich tissue. In the monocotyledon, Avena sativa, and perhaps monocotyledons in general, decay rate is maximal at a low P_FR concentration and the decay curve is the same under continuous red, incandescent and blue light. This dicotyledon/monocotyledon difference with respect to saturation of phytochrome decay does not correlate with the other dicotyledon/monocotyledon difference, the presence or absence of dark reverions of P_FR to P_R, since the dicotyledons Amaranthus and Mirabilis that lack reversion still show no saturation of decay. Possible growth control by the P_FR/P_total ratio is discussed in relation to environmental changes in light quality.Research carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission.     

Investigations on the role of ethylene in phytochrome-mediated photomorphogenesis

The etiolating, intact mustard (Sinapis alba L.) seedling exhibits a distinct temporal pattern of ethylene production. Light, operating through phytochrome, increases the rate of ethylene production without changing the pattern. Ethylene production of the isolated plant parts (segments), added together, exceed the production of the intact system even if the wound effect is taken into account. There is no significant light effect on ethylene production of the segments. Phytochrome-mediated anthocyanin synthesis in the cotyledons is inhibited by ethylene. The responsiveness towards ethylene of the anthocyanin producing metabolic chain is decreased by phytochrome. As anthocyanin synthesis is only partly inhibited under saturating ethylene concentrations in the atmosphere around the seedlings (100 l l^–1), a twofactor analysis becomes feasible. This analysis leads to the result that phytochrome and ethylene show multiplicative behavior, meaning that phytochrome and ethylene act on the same metabolic sequence (leading to anthocyanin) but independently of each other, and at different sites. Therefore, the hypothesis that ethylene mediates the action of phytochrome in anthocyanin synthesis and photomorphogenesis in general appears to be inapplicable.Abbreviations P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome, i.e. [P_r]+[P_fr]     

Modification of apparent phytochrome synthesis in pisum by inhibitors and growth regulators

The repeated exposure of Pisum (pea) plants to red light brings into operation an apparent synthesis of phytochrome which is not observed in material kept in the dark. This process shows some temperature compensation but has an optimum at 26°; it is irreversibly inhibited by 10⁻⁴ m cycloheximide and 10 μg/ml actinomycin D. It is also inhibited by the auxins indoleacetic acid, naphthalene acetic acid and 2,4-dichlorophenoxyacetic acid at 10⁻⁴ m but in these cases the inhibition is completely reversed when the auxin is washed out of the tissue. Antiauxins 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxy isobutyric acid, while strongly inhibiting growth have little effect on apparent synthesis. Other growth regulators and the precursor of tetrapyrrole synthesis, δ-aminolevulinic acid, have no consistent effect on the process, but 3 × 10⁻⁴ m cobalt (II) nitrate is inhibitory. The capacity for apparent synthesis decreases as the cells approach maturity. The results may be explained by either de novo synthesis of phytochrome, or by a transformation process resembling in some respects the dark reversion of Pfr to Pr. The physiological role of apparent synthesis is suggested.     

Etude du phytochrome detectable,in vivo dans les graines de Cucurbita pepo L. au cours des differentes phases de la germination

Summary In dry gourd seeds all the phytochrome is in the P_fr form. The increase of phytochrome content from the beginning of hydration involves two phases, A and B, in the embryonic axis as well as in the cotyledons. Cycloheximide does not prevent the appearance of P_r during phase A. We assume that P_r is gradually released from an inactive complex. On the other hand phase B is inhibited by cycloheximide; this could mean that a de novo synthesis of P_r occurs.Some experiments indicate that the phytochrome which is localized in the embryonic axis may be involved only in the germinating process.The phytochrome which is synthesized during phase B disappears when the seeds are irradiated with red light, while the original phytochrome does not.According to our data it seems necessary to lay down a new and precise definition of the germination process.     

Relationships between phytochrome state and photosensitive growth of Avena coleoptile segments

Using various photostationary state light sources to obtain reproducible phytochrome conversion of from 5 to 88% P_FR, assayed by 2 wavelength in vivo spectrophotometry, relationships between initial percent P_FR and elongation of apical Avena coleoptile segments over the succeeding 20 hours in darkness were studied. With material grown in total darkness, all P_FR levels promote elongation, and maximal promotion requires roughly 50% P_FR. The promotion caused by an initial 5 minute red (88% P_FR) treatment at hour 0 is partially reversible at hour 5 by sources forming less than 48% P_FR, but totally irreversible at hour 8, though less than 50% of the growth has been accomplished by this time. Direct photometric assays at hour 5 indicate a phytochrome state of roughly 45% P_FR, consistent with the reversal data. At hour 8, however, 11 to 22% of the phytochrome still assays as P_FR, an inconsistency suggesting simply that the elongation process has proceeded beyond photochemical control. Thus, in contrast with results previously reported for Pisum and Phaseolus, there is no contradiction between photometric and physiological assays of phytochrome state in Avena coleoptile segments.

Attempts to expand this study by using segments from seedlings pretreated with red light showed that such pretreatment as little as 1 to 2 hours before drastically reduces subsequent elongation and photoresponse on the medium employed. This decline in growth potential can be halted at any time before its completion by either excision of the segment or far-red treatment of the intact seedling.

     

The biosynthesis of alkaline phosphatase with a particulate fraction of Escherichia coli. The mechanism of inhibition by inorganic phosphate

1. By digitonin lysis of penicillin spheroplasts of Escherichia coli a particulate fraction P₁ was previously obtained that supported the sustained synthesis of alkaline phosphatase when supplied with amino acids, nucleotide triphosphates and other cofactors. This P₁ fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg²⁺, yielded the P₁(S) fraction, consisting of integrated particulate subcellular particles containing DNA and RNA. 2. The P₁(S) fraction from E. coli K10 wild type (R⁺₁R⁺₂P⁺) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in vitro. The synthesis occurred in phases. The first was followed by a lag, and then there was a linear rapid phase that continued for at least 3hr. Actinomycin D inhibited the appearance of the second phase. It was concluded that the particles are programmed to synthesize enzyme even when prepared from repressed cells, and therefore that synthesis of the specific messenger RNA for alkaline phosphatase in vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. 3. Phosphate inhibited synthesis of enzyme to the same extent with the P₁(S) fractions of two constitutive strains as with the P₁(S) fraction of the wild-type strain. 4. Inorganic phosphate inhibited amino acid incorporation with the P₁(S) fraction and also inhibited enzyme synthesis in vitro. The effect on amino acid incorporation could be partially overcome by adding Mn²⁺ to the incubation mixtures. However, Mn²⁺ inhibited the synthesis of alkaline phosphatase. Also, inhibition of the incorporation of [³²P]CTP into RNA was overcome by Mn²⁺. The effect of phosphate on amino acid uptake was most probably due to a phosphorolysis of RNA by polynucleotide phosphorylase, also present in the P₁(S) fraction. This phosphorolysis may be responsible for the instability of messenger RNA in vitro and in vivo. 5. Phosphate also specifically inhibited the formation of alkaline phosphatase, since it did not affect markedly the induced formation of β-galactosidase by the same P₁(S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn²⁺-dependent reaction. It is suggested that the repression of the synthesis of alkaline phosphatase in vivo in the wild-type strain was the sum of these two effects.     

Response of tissue with different phytochrome contents to various initial photostationary States

Pretreatment of etiolated pea plants with red light and with red combined with far-red light produced morphologically similar plants having 4-fold differences in spectrophotometrically detectable phytochrome. Stem segments from the variously pretreated plants respond in the same way to different percentage conversions of phytochrome to P_FR. These results suggest that the P_FR./P_R ratio, rather than the concentration of P_FR, governs pea stem segment elongation. However, the ratio hypothesis does not explain contradictions between spectrophotometric and physiological assays previously obtained with this tissue, nor does it explain similar contradictions obtained in other systems. The only hypothesis consistent with the data to date is that of the existence of bulk and active phytochrome fractions, with the latter present in insufficient quantities to be spectrophotometrically detectable.     

Phytochrome changes correlated to mesocotyl inhibition in etiolated Avena seedlings

The elongation of etiolated Avena mesocotyls is inhibited by red light (660 mμ). Immediately after exposing mesocotyl sections to varying doses of red light the ensuing concentrations of phytochrome in the far-red absorbing form (P₇₃₀) were measured. The extent of mesocotyl inhibition observed 5 days later is proportional to the logarithm of P₇₃₀ concentration in mesocotyl tissue at the time of red light exposure.

The inhibition of mesocotyl growth by red light can be reversed partially by subsequent exposure to far-red light (730 mμ). Increasing doses of far-red light result in decreasing concentrations of P₇₃₀ as compared with the original P₇₃₀ level due to the preceding red light exposure. The reduced mesocotyl inhibition of seedings which had been exposed to red and far-red light is proportional to the logarithm of P₇₃₀ concentration remaining in the tissue at the end of the two light exposures.

This indicates that the same correlation exists between P₇₃₀ concentration and growth response whether the seedlings had been exposed to red light only or to red followed by far-red light.

     

Induction versus modulation in phytochrome-regulated biochemical processes

The time course of appearance of competence towards phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does phytochrome act to turn responses on (induction), or — as an alternative — does phytochrome cause an amplification of processes already occurring in absolute darkness albeit at low rates once competence is reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25° C. In the case of anthocyanin, it was found that two distinct points of competence exist (26 h and 39 h after sowing, 25° C). In the case of ‘early anthocyanin’ (competence point at 26 h), synthesis does not occur in darkness without P_fr, while in the case of ‘late anthocyanin’ (competence point at 39 h), phytochrome causes an amplification of a process occurring in complete darkness albeit at a very low rate. It is concluded that in phytochrome-mediated photomorphogenesis, modulation as well as induction of biosynthetic processes plays a role.     

Some regulatory properties of purine biosynthesis de novo in long-term cultures of epithelial-like rat liver cells

De novo synthesis of purine nucleotides and some regulatory properties of this pathway were studied in cultured epithelial-like rat liver cells. It was found that the physiological 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) concentration in these cells is limiting for purine synthesis de novo. Increase of P-Rib-PP availability, achieved by activation of P-Rib-PP synthetase at high P_i concentration, resulted in acceleration of purine synthesis. The effects of increasing cellular ribose 5-phosphate (Rib-5-P availability, by methylene blue-induced acceleration of the oxidative pentose phosphate pathway, on P-Rib-PP availability and on the rate of the novo purine synthesis were also studied. It was found that at the P_i concentration prevailing in the tissue at extracellular physiological P_i concentration, Rib-5-P availability is saturating for P-Rib-PP generation and therefore also for purine synthesis.     

Stability of phytochrome concentration in dicotyledonous tissues under continuous far-red light

Summary The phytochrome concentration in dark-grown seedlings of Pisum sativum, Phaseolus aureus and Sinapis alba remained constant under continuous far-red illumination for periods of up to 6 hours. Similar treatment of Zea mays seedlings reduced the phytochrome concentration by more than 60 percent. The results in the dicotyledonous seedlings may be due to the reversion of P_fr to P_r at a rate sufficient to prevent P_fr destruction; no evidence for reversion has been detected in Zea. Typical photomorphogenic responses were observed in the dicotyledonous seedlings in the absence of P_fr destruction.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome