INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE REDUCTASE ACTIVITIES IN MAIZE

Abstract— Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety 'Ganga-5' is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO^-₃, was saturated, further increased both enzyme activities. Even if red light was given 4–8 h before NO^-₃, it still increased both NR and NIR activities.     

FLUENCE RATE COMPENSATION OF THE PERCEPTION OF RED: FAR-RED RATIO BY PHYTOCHROME IN LIGHT-GROWN SEEDLINGS*

Abstract— The hypothesis that phytochrome functions as a sensor of vegetational shade through the perception of the red: far-red photon fluence rate ratio requires that the mechanism of perception be compensated for wavelength-independent fluctuations in fluence rate (Smith, 1982). This paper seeks to establish the lower limit of fluence-rate compensation and to assess whether or not compensation is effective at the total fluence rates typical of herbaceous canopies. Using specially-designed cabinets, Sinapis alba L. (white mustard) seedlings were grown from germination under a range of total photosynthetically-active radiation (PAR = 400 to 700 nm) values and a range of red: far-red ratios. The data indicate that fluence-rate compensation is effective above a PAR value of ca. 60 μ.mol m² s'. Pretreating seedlings at high red: far-red ratio and a PAR level of 300 (μmol m₂S_-1for increasing periods of time led to an extension of fluence rate compensation to lower fluence rates. The results are discussed in relation to the photosynthetic competence of the seedlings grown under these conditions.     

REGULATION OF NITRATE REDUCTASE ACTIVITY IN RICE

The properties of NR from rice varieties Yuanfengzao and Jingyin 127 are the same in molecular weight of native enzyme and subunit, optimum pH, K_m value for substrates NO_3~- and NADH, stability in vitro and immunological characteristics. The decay rate in vitro of the activity of the partially purified NR from Yuanfengzao is 20％ lower than that from Jingyin 127. The results from antiserum titration and rocket immunoelectrophoresis indicated that the amount of NR in Yuanfengzao is about 1.5 times more than that in Jingyin 127. Further study on in vivo incorporation of ~3H-labeled amino acids into NR revealed that the rate of NR synthesis in Yuanfengzao is obviously higher than that in Jingyin 127. In vitro translation of mRNA showed that the products of NR-mRNA per unit of total mRNA of Yuanfengzao is much more than that of Jingyin 127. It is concluded that, therefore, NR activity in rice is regulated through NR synthesis and NR-inhibitory protein(s) system, the former is further controlled at the lev     

CONTROL BY PHYTOCHROME OF CHLOROPHYLL SYNTHESIS IN SEEDLINGS OF SORGHUM VULGARE

The strong effect of light pretreatments on the synthesis of chlorophyll-a and-b in the shoot of Sorghum vulgare (kept under saturating white light) can be attributed to phytochrome only. No specific blue light effect was found. The phytochrome system appears to function perfectly normally under these conditions. Escape from reversibility is not detectable up to approximately 40 min after the onset of an inductive red light pulse. Thereafter, escape is fast, being completed at approximately 2.5 h after the inductive light pulse.     

PHYTOCHROME IN GREEN-TISSUE: PARTIAL PURIFICATION and CHARACTERIZATION OF THE 118-KILODALTON PHYTOCHROME SPECIES FROM LIGHT-GROWN Avena sativa L.*

The predominant, immunochemically-detectable phytochrome polypeptide rapidly extracted directly into boiling sodium dodecyl sulfate-containing buffer from fresh or freeze-dried green Avena tissue has an apparent molecular mass of 118 kilodaltons (kDa). This result indicates that the 118-kDa phytochrome species obtained from green Avena by extraction and rapid processing under non-denaturing conditions in previous studies was not derived by partial proteolysis of a larger polypeptide present in the cell. Additional data do, however, demonstrate the presence in green tissue homogenates of proteolytic activity that can cause a = 6-kDa reduction in apparent molecular mass and a blue-shift in the P_fr absorbance maximum of phytochrome during handling. This proteolytic activity contrasts with that previously encountered in etiolated tissue in that it is not inhibited by phenylmethylsulfonyl fluoride, but is inhibited by iodoacetamide and leupeptin. This result indicates that the activity is associated with a thiol-like protease. A partial purification procedure that incorporates the use of iodacetamide and a novel chromatographic step is described for green-tissue phytochrome. This procedure provides 50% recovery with a 90-fold enrichment of phytochrome relative to the initial extract in which the chromoprotein is 0.003% of the total soluble protein. The final fraction is apparently free of proteolytic activity. Immunoblot analysis of this fraction demonstrates that the predominant immunoreactive band has a monomeric molecular mass of 118 kDa. Comigration of this band with a band exhibiting zinc-induced fluorescence on blots of the partially purified preparations verifies that the 118-kDa species is the principal tetrapyrrole-bearing polypeptide present. Spectral properties of the final fraction are identical to those published for crude green-tissue extracts, indicating the stability of the molecule's spectral properties throughout the procedure. Size exclusion chromatography under nondenaturing conditions shows that the 118-kDa phytochrome species from green tissue comigrates with the dimeric, etiolated-tissue molecule, and is therefore suggestive of similar quaternary structure. Together these data reinforce previous conclusions that the predominant phytochrome molecule present in the living cells of green tissue is resolvable as a 118-kDa species, distinct from the well-characterized 124-kDa molecule from etiolated tissue (Tokuhisa et al., 1985, Planta 164, 321–332), and indicate that the partial purification protocol described here sustains the green-tissue phytochrome in its native state throughout the procedure.     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

CONTROL BY PHYTOCHROME OF EXTENSION GROWTH and POLAROTROPISM IN CHLORONEMATA OF Funaria hygrometrica

Abstract— The photocontrol of extension growth and polarotropism has been investigated in chloronemata of the moss Funaria hygrometrica Hedw. When grown on a 12/12 h light/dark cycle, chloronemata show light-stimulated diurnal variations in elongation rate with no evidence of a circadian rhythm. Regulation of elongation by a low energy, photoreversible phytochrome mechanism was demonstrated by brief red (R) and far red (FR) irradiations given either at the end of the day (EOD) or 6 h later as a night break (NB). Night break irradiation with polarised R caused polarotropic reorientations of chloronemal growth also through a FR-reversible, low energy phytochrome mechanism. Such transformations of P_r to P_fr were accompanied by a 90° shift in the orientation of the phytochrome chromophore. Microbeam irradiation indicated that the phytochrome chromophores involved in both responses were predominantly, if not entirely, located in the tip of the apical chloronemal cell.     

PHYTOCHROME DESTRUCTION IN DARK - AND LIGHT-GROWN AMARANTHUS CAUDATUS SEEDLINGS

Abstract— Slow destruction of the far-red-absorbing form of phytochrome (P_fr), which has been observed in light-grown oat and maize, occurs in light- and dark-grown Amaranthus, Pharbitis , and Brassica seedlings as well. Destruction of P_fr in these seedlings shows two phases: if a high level of P_fr is produced in dark-grown seedlings, the destruction is fast in the beginning and then slows after a low P_fr level has been reached. Slow P_fr destruction is predominant in light-grown tissue.     

BLUE LIGHT RESPONSES IN NITRATE REDUCTASE MUTANTS OF NEUROSPORA CRASSA

Abstract— The role of nitrate reductase in the blue light responses of photosuppression and phase shifting of circadian conidiation was studied in Neurospora crassa. The photoresponses, as assayed in three nitrate reductase mutants (nit-1,nit–2 and nit-3), showed no significant differences as compared to the responses in a strain which could utilize nitrate. In addition, the responses occur on a medium which (i) represses nitrate reductase activity due to the presence of ammonium ion and arginine or (ii) results in the production of an inactive enzyme due to the presence of tungsten. Nitrate reductase appears to be of no or secondary importance as a photoreceptor in the responses studied.     

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.     

A POSSIBLE CONTROL BY PHYTOCHROME and OTHER PHOTORECEPTORS OF PROTEIN ACCUMULATION IN THE GREEN ALGA Ulva rigida

Abstract— The effects of red, blue and green light pulses on total protein accumulation in the green alga Ulva rigida following transfer from a low to high nitrate medium in darkness were examined. Red light pulses prior to transfer to darkness increased protein accumulation by about 55%. Blue and green light pulses also stimulated protein accumulation, but to a lesser extent (40-30% respectively). Stimulation of protein accumulation by red, blue or green light was largely (red light) or partially (blue or green light) reversible by far-red. The role of phytochrome and a red/green photoreversible system in the control of protein synthesis is discussed.     

IRREVERSIBLE PHOTO IN ACTIVATION OF NITRATE REDUCTASE DURING THE FLAVIN-SENSITIZED PHOTOCHEMICAL ASSAY OF ITS CATALYTIC ACTIVITY

Abstract— The classic photochemical system in which flavin and EDTA act as photosensitizer and electron donor, respectively, has been employed for assaying in vitro the catalytic activity of Ankistrodesmus braunii nitrate reductase. When the photochemical assay is performed under air, but not in anaerobiosis, a considerable decay in the nitrite photoproduction rate is observed after 10–15 min. which is accompanied by a decrease of reduced methyl viologen-nitrate reductase activity. The first enzyme activity, i.e. diaphorase is photoinactivated even more quickly and does not present any initial lag phase. Some oxygen species (superoxide and/or hydrogen peroxide) are probably involved in the photoinactivating mechanism, which appears to be non-specific and irreversible.
The use of flavin/EDTA photosystems is therefore, very practical for the in vitro assay of nitrate reductase activity, although anaerobic conditions are required for optimum results. Since inactivation of the terminal activity is, however, relatively slow, anaerobiosis would not be required for assay times shorter than 10 min.     

PHOTOACTIVATION OF NITRATE REDUCTASE FROM NEUROSPORA CRASSA

Abstract— The photoactivation of nitrate reductase from Neurospora crassa was studied in partially purified extracts. The inactive enzyme [inactivated by reduction in the presence of potassium cyanide] could be reactivated by chemical oxidation with ferricyanide or by irradiation with blue light. The enzyme contains a short electron transfer chain consisting of flavin adenine dinucleotide, cytochrome b ₅₅₇ and molybdenum which normally transfers electrons from reduced pyridine nucleotide to nitrate. This overall activity, which was negligible in the inactive enzyme, was restored to approximately 70% of the ferricyanide control by irradiation. However, nitrate reduction using reduced methylviologen as reducing power, which was also negligible in the inactive enzyme, was photoactivated to 100%. The diaphorase activity of the enzyme mediated by the flavin adenine dinucleotide, which was fully active in the inactivated enzyme, was inhibited approximately 30% by the irradiation treatment. The action spectrum for photoactivation showed that a flavin was the photoreceptor chromophore. Photoactivation occurs only in the presence of oxygen.     

APPEARANCE OF PHOTOPHOSPHORYLATION CAPACITY: THRESHOLD vs GRADED CONTROL BY PHYTOCHROME

Abstract— It is shown that in attached mustard cotyledons graded control of chlorophyll synthesis by physiologically active phytochrome (P_fr) and threshold control by P_fr of the 'potential capacity' to photophosphorylate are totally different phytochrome actions even though both controls are essential for the build-up of the same functional complex, the machinery for photophosphorylation. The essential findings are as follows: The action of P_fr (made by a 1 min red light pulse) on the capacity and efficiency of photophosphorylation is rapid—detectable after 15 min and completed after 30 min—whereas the action of P_fr on chlorophyll formation is slower—only detectable 45 min after the original red light pulse (R). Detailed escape studies (loss of full reversibility of the inductive effect of a R pulse by far-red) show that the effect of a R pulse on chlorophyll synthesis remains fully reversible for 45 min whereas the action of P_fr on the capacity for photophosphorylation is very fast (occurring within 2 min). Control of capacity for photophosphorylation is a threshold response (whereby the threshold value is approximately 1.25% P_fr based on total phytochrome at 36 h = 100%) whereas control by P_fr of chlorophyll synthesis is graded. Control of capacity for photophosphorylation by P_fr only operates if the hypocotyl hook is connected to the cotyledons for at least 2 min after the inductive R pulse, i.e. until full escape from reversibility has occurred, whereas chlorophyll formation in the cotyledons is not affected by the separation of hook and cotyledons.     

PREVENTION BY PHYTOCHROME OF PHOTODELAY IN CHLOROPHYLL ACCUMULATION

A photodelay in chlorophyll- a and -b accumulation is observed in mustard ( Sinapis alba L.) cotyledons during the first 3 h after the onset of white light even at medium light fluxes (3500 lx). A pretreatment of two red light pulses or a 12 h far-red light pretreatment, both operating through phytochrome. prevent the photodelay completely. This is a specific phytochrome effect since'it can be separated from the effects of phytochrome on the rates during pre-steady state and steady state phases of chlorophyll accumulation in saturating white light. Thus, photostability of chlorophyll in nature is a photoresponse mediated through phytochrome.     

THE ROLE OF HYDRATION IN THE PHOTOTRANSFORMATION OF PHYTOCHROME

Abstract— Changes in the visible absorption spectrum and in phototransformation of phytochrome have been found to be associated with dehydration and rehydration. These spectral properties explain the previously reported rapid appearance of phytochrome in imbibing seeds. No evidence was found for the orientation of the phytochrome chromophore in gelatin films or in preparations subjected to a shearing force.     

REGULATION OF PROTEIN PHOSPHORYLATION BY PHYTOCHROME IN Sorghum bicolor

Abstract— In vitro phosphorylation of some polypeptides was affected in extracts obtained from 5-and 6-day-old plants irradiated with 5 min of red light. The phosphorylation of 55 kDa polypeptide in both 5- and 6-day-old plants, a 60 kDa, and 76 kDa polypeptide in 6-day-old plants and 70 kDa, 67 kDa polypeptide in 5-day-old plants was stimulated by red light. This effect was reversible by far-red light. The extent of stimulation by red light and reversal by far-red light varied for different polypeptides. No differential effect of red and far-red light was seen on the phosphorylation of 94 and 40 kDa polypeptides. In fact, phosphorylation of 94 kDa polypeptide in 6-day-old plants decreased on red light irradiation. These results show that the phosphorylation or dephosphorylation of some proteins is affected by phytochrome and the effect of light is also dependent on the age of the plant.     

THE FUNCTION OF PHYTOCHROME IN THE NATURAL ENVIRONMENT—III. MEASUREMENT AND CALCULATION OF PHYTOCHROME PHOTOEQUILIBRIA

Abstract— Phytochrome photoequilibria have been measured in dark-grown Phaseolus uulgaris L . and Cucurbita pepo I . hypocotyl hooks which had been exposed to various natural and artificial radiation sources. Mean phytochrome photoequilibria ( φ ) varied from 0.20 within a wheat canopy to 0.54 above, although lower values were occasionally observed in densely shaded areas. Greater variation in phyto chrome photoequilibria and lower levels of P_fr were recorded within a sugar beet canopy. The range of photoequilibria was φ= 0.04 in dense shade to φ= 0.54 above the canopy. Photoequilibrium was achieved within 5 s in mid-day sunlight and approximately 30 s in dense canopy shade.
A close correlation was found between φ and the ratio of the quantum flux in the red and far-red wavelength bands (ζ) in broad spectrum (400–800 nm) radiation. This relationship allows direct prediction of φ from a knowledge of ζ. Phytochrome showed greatest sensitivity to spectral changes in the range ζ= zero to ζ= 1.0, which is the range found in the natural environment.
The observations provide support for the hypothesis that phytochrome is involved in the detection of shading by plants.