PHYTOCHROME-STIMULATED REGENERATION OF PROTOCHLOROPHYLL IN COTYLEDONS OF THE MUSTARD SEEDLING

Abstract —It has been shown [Kasemir, H., U. Oberdorfer and H. Mohr, Photochem. Photobiol. (1973) 18 , 481–486] that elimination of the lag phase of chlorophyll a (Chl) accumulation in continuous white light is due exclusively to the action of phytochrome (P_fr). In the present paper we show that the action of P_fr on the lag phase of Chl accumulation can be understood quantitatively as a consequence of the action of P_fr on the initial rate of protochlorophyll (PChl) regeneration. Disappearance of PChl (or formation of Chl) can be excluded as a control signal for the light-mediated changes in rate of PChl regeneration. The P_fr control of PChl regeneration does not discriminate between PChl 650 and PChl 637. The action of P_fr on the PChl regeneration is a relatively fast process (time lag < 3 h). On the other hand, the effect remains stable over long periods (at least 24 h) in darkness.     

CHANGES IN THE RATES OF PHOTOCONVERSION OF PHYTOCHROME DURING ETIOLATION IN MUSTARD SEEDLINGS

Abstract— The relative phytochrome photoconversion rates in cotyledons and hypocotylar hook of etiolating mustard ( Sinapis alba L.) seedlings were measured between 16 and 96 h after sowing. It was found that at constant fluence rates photoconversion rate in red light increases in both organs with time whereas the photoconversion rate in far-red (756 nm) light decreases with time of development. Since the isosbestic point remains constant, it was concluded that the observed changes cannot be attributed to changes of extinction coefficients. It was not possible, however, to decide whether the observed changes are due to changes of light attenuation or quantum yields.     

PHYSIOLOGICAL RELATIONSHIPS BETWEEN PHYTOCHROME EFFECTS ON INTERNODE EXTENSION GROWTH AND DRY MATTER ACCUMULATION INLIGHT-GROWN MUSTARD

The physiological relationships between the effects of phytochrome photoequilibrium (Pfr/P) on internode extension growth and dry matter accumulation were investigated in white light (WL)-grown Sinapis alba L. seedlings. After 11 days under continuous WL, the seedlings were exposed: (a) to pulses of light providing different Pfr/P, followed by 24 h darkness (D); (b) to pulses of light providing different Pfr/P, followed by 3 h D and 24 h continuous WL; (c) to continuous WL with or without supplementary far-red light (to reduce Pfr/P); or (d) to pulses of light providing different Pfr/P followed by D, in factorial combination with either water or a saturating (0.2 M) sucrose solution applied to one of the leaves. In D (“a” and “d”) low, compared to high Pfr/P increased both internode extension growth and dry weight to the same extent. Under WL (“b” and “c”) low PfrlP promoted internode extension growth but had no proportional effects on internode dry weight. Sucrose promoted internode extension growth with a lag of at least 8 h (compared to the rapid effect of low Pfr/P) and did not reduce the effect of low Pfr/P. These results indicate that Pfr/P effects on internode extension growth are not the consequence of changes in photoassimilate translocation from the leaves. Under WL, PfdP effects on internode length occur partially at the expense of internode dry matter per unit length.     

INTERORGAN CORRELATION IN A PHYTOCHROME-MEDIATED RESPONSE IN THE MUSTARD SEEDLING

Abstract —Phytochrome ( P_fr )-mediated threshold control of apparent lipoxygenase (LOG) synthesis in the cotyledons of the mustard seedling depends on the presence of the hypocotylar hook. With isolated cotyledons, no suppression by light of apparent LOG synthesis can be detected. As soon as the cotyledons are separated from the hook, the control of apparent LOG synthesis by P_fr is completely lost. The data are consistent with the hypothesis advanced in a previous paper [Oelze-Karow, H. and H. Mohr (1973) Photochem. Photobiol. 18, 319–330] that apparent LOG synthesis in the cotyledons is controlled by phytochrome located in the hypocotylar hook. The data support the concept [De Greef, J. A. and R. Caubergs (1972) Arch. Int. Physiol. Biochim. 80, 959–960] that a very precise and highly ordered biophysical recognition and transmission system of light signals exists in plants.     

THE DOSE RESPONSE CURVE IN PHYTOCHROME-MEDIATED ANTHOCYANIN SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —The dose response curve for light (phytochrome)-induced anthocyanin synthesis was determined in the mustard seedling. The curve gives the amount of anthocyanin (A) synthesized within 24 h as a function of the amount of P_fr* produced by a brief light pulse. The [P_fr] response curve is composed of two linear parts with very different slopes ( a _1,2) connected by a relatively narrow transient range (curved segment). The [P_fr] response curve extrapolates precisely through zero [P_fr]. The reciprocity law is valid over the whole range investigated (up to 320 s of irradiation). It is concluded that the initial (or primary) reaction of P_fr (P_fr+ X → P_frX) does not involve any significant cooperativity in the case of phytochrome-mediated anthocyanin synthesis. It is speculated that the linear parts of the [P_fr] response curve truly reflect the mode of phytochrome action ( A = a _1,2 [P_fr]; X does not come into play since it is not rate limiting) whereas the curved segment represents a transition of the reaction matrix of P_fr. The large difference between a₁ and a₂ seems to indicate that the physiological effectiveness of a given amount of P_fr (or P_frX) is determined by [P_fr] through a P_fr-induced change in the reaction matrix.     

AN ATTEMPT TO LOCALIZE THE THRESHOLD REACTION IN PHYTOCHROME-MEDIATED CONTROL OF LIPOXYGENASE SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —Synthesis* of the enzyme lipoxygenase (LOG)? in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr) through a threshold (all-or-none) mechanism. The data of the present paper confirm the previous assumption (Oelze-Karow and Mohr, 1973) that the primary reaction of P_fr (P_fr+ X → P_frX ? P_frX‘) is the site of the highly cooperative threshold reaction. Suppression of LOG synthesis depends on the presence of P_frX’. However, P_frX‘ is only stable above the threshold level of P_fr. If the level of P_fr is below the threshold, P_frX is stable, and no suppression of LOG synthesis occurs. As long as the level of P_fr remains below the threshold, no destruction of P_fr takes place. Destruction of P_fr occurs only as long as [P_fr]?is above the threshold level. Thus the simplest formulation of the actual threshold reaction in the LOG response is P_frX?_frX’ state at [P_fr] below threshold no P_fr destruction LOG synthesis suppressed state at [P_fr] above threshold P_fr destruction(¹k_d LOG synthesis unimpaired The reversible threshold reaction is thus an integral part of the “primary reaction” of P_fr occurring at the “matrix” specific for the LOG response. The data and conclusions on the LOG response are consistent with an “open phytochrome-receptor model” recently advanced by E. Schäfer (1975). The data are not consistent with the concept that a rapid dark reversion (P_fr→P_r) exists in dicotyledonous seedlings and that the degree of P_fr dark reversion strongly depends on the initial photostationary state, φ?, established by a saturating light pulse.     

STIMULATION OF THE SHIBATA SHIFT BY PHOTOCHROME IN THE COTYLEDONS OF THE MUSTARD SEEDLING SINAPIS ALBA L.

Abstract— In the cotyledons of the mustard seedling Sinapis alba L. the duration of the Shibata shift can be greatly shortened by a pretreatment with light pulses prior to the protochlorophyllide– chloro-phyllide a photoconversion. It was shown that the light pulses act through photochrome (P _fr ). Since reversibility of a red light pulse induction by a far-red light pulse is rapidly lost (within 2 min) it is concluded that at least the initial action of P_fr occurs rapidly in this response. On the other hand, the effect of a red light pulse on the rate of protochlorophyll regeneration in the mustard seedling cotyledons is fully reversible by a far-red light pulse for more than 5 min. It is concluded that control of protochlorophyll regeneration and control of the Shibata shift by phytochrome cannot be consequences of the same initial action of P_fr Apparently P_fr controls both phenomena independently.     

VARIATION IN THE RATES OF SYNTHESIS AND DEGRADATION OF PHYTOCHROME IN COTYLEDONS OF CUCURBITA PEPO L. DURING SEEDLING DEVELOPMENT

Abstract— The time courses for P_r appearance, P_r disappearance and P_fr destruction have been analysed in cotyledons of Cucurbita pepo L. after different preirradiation programs. In etiolated seedlings the rate of P_r appearance is low in young seedlings reaching a maximum in 3.5–5 day old seedlings then decreasing rapidly with increasing age. The rate of P_fr destruction is very low in young seedlings, increases rapidly up to the 4th day and then remains almost constant. The disappearance of P_r becomes significant for seedlings older than 45 days. These reactions seem not to be influenced by short preirradiations. However, after prolonged preirradiation, a degree of control of P, appearance and/or disappearance by the "internal clock appears to be operative.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.     

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.     

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.     

PHYTOCHROME CONTROL OF EXTRACELLULAR PEROXIDASE ACTIVITY IN MUSTARD INTERNODES: CORRELATION WITH GROWTH, and COMPARISON WITH THE EFFECT OF WOUNDING

Abstract— The effects of phytochrome status on extracellular peroxidase activity were investigated in Sinapis alba L. seedlings grown for 12 days under continuous white light and transferred to darkness after a red light or a far-red light pulse. The rates of extension growth and dry matter accumulation in the first internode were increased by the far-red light pulse. Extracellular proteins, obtained by low speed centrifugation of intact internodes infiltrated with CaCl₂, were separated by isoelectrofocusing, and four extracellular acidic peroxidases were resolved, the most active being A3 and A4 (both ˜60 kD). The activity of A4 was reduced by the far-red light pulse perceived by phytochrome, while the activity of A3 was unaffected. The promotion of internode extension growth caused by far-red light is biphasic [Casal and Smith (1989) Plant, Cell Environ. 12 ,511–50]. Changes in peroxidase activity were detected prior to the second, but not to the first phase of the internode growth promotion. The effects on both growth and peroxidase activity were virtually restricted to the upper half of the internode and, once established, did not subsequently increase in magnitude.
In contrast to the effects mediated by phytochrome, blue light pretreatments affected growth but not extracellular peroxidase activity. Wounding the internode reduced extension growth, increased the activity of A3, but caused no significant effects on A4.
Other extracellular proteins, separated in sodium dodecyl sulphate polyacrylamide gels and stained with Coomassie blue, showed no significant differences. The concentration of extracellular proteins was higher in the upper than the lower half of the internode.
Results are discussed in terms of phytochrome effects in light grown plants, peroxidase activitv-arowth relationships, and extracellular peroxidase isoform functions.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

THE EFFECT OF PROLONGED LIGHT EXPOSURE ON THE EFFECTIVENESS OF PHYTOCHROME IN ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS*

Abstract— The hypocotyl of the tomato ( Lycopersicon esculentum ) seedling synthesizes large amounts of anthocyanin if exposed to prolonged light. Single light pulses are totally ineffective. The involvement of phytochrome can be shown by light pulse treatments following a prolonged light exposure. It is predominantly the action of blue/UV light which leads to a high responsiveness of anthocyanin synthesis towards phytochrome. Moreover, the data suggest a phytochrome-independent action of blue/UV light, in particular of UV-B, on anthocyanin synthesis.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

EFFECT OF PHYTOCHROME ON THE BIOSYNTHESIS OF ACYCLIC AND CYCLIC CAROTENOIDS IN HIGHER PLANTS

Radish plants were grown in the presence of three different herbicides that interfere with the formation of the normal range of cyclic carotenoids, leading to an accumulation of acyclic biosynthetic intermediates, mainly phytoene (SAN 6706 and amitrole) and zeta-carotene (3852). Plants were then irradiated by four different light programs in order to gain more insight into the first steps of carotenoid biosynthesis and their control by light and phytochrome. In all cases, herbicide-treated and control, carotenoid biosynthesis was greatly enhanced by red light consistent with an effect of phytochrome on the early steps of the pathway. However, similar enhancement was also obtained after treatment with far-red light. Indeed with SAN 6706-treated plants synthesis of phytoene was stimulated to a much greater extent by far-red light given alone, than by red light. The involvement of phytochrome in the regulation of carotenoid biosynthesis appears not to be as simple as previously supposed.     

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.     

ON THE POLARITY OF PHYTOCHROME CONTROLLED LEAF UNFOLDING IN DARK-GROWN SEEDLINGS OF TRITICUM AESTIVUM*

Abstract— The apical ends of sections cut from etiolated primary wheat leaves show a higher degree of phytochrome-mediated leaf unfolding than the basal ends, regardless of the position of the segment in the leaf. It could be clarified that this polarity is not the result of a stable gradient within the intact dark grown leaf. Partial irradiation experiments have shown that leaf unfolding can be induced much more effectively by an irradiation of the apical half of a section than of the basal half. Though only the basal end of the sections was incubated with gibberellic acid (GA₃) in darkness, the degree of the GA₃-induced leaf unfolding was highest at the apical end of these non-irradiated segments. The results suggest that in each separate leaf section a new gradient of a still unknown “unfolding-factor” is established.