PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De-etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far-red absorbing form of phytochrome (P_fr) to receptors essential for chlorophyll synthesis, thereby increasing the local P_fr/total phytochrome (P_tot)ratio. We have studied etioplasts as possible loci for such P_(r receptors. The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic P_fr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model. Dark destruction of bulk spectrophotometric P_fr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk P_fr is complete. Since this demonstrates persistent presence of P_fr active in potentiation, we propose that at least part of this P_fr is associated with the etioplasts.     

RHYTHMIC REGULATION OF THE LIGHT-HARVESTING CHLOROPHYLL alb PROTEIN and THE SMALL SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE mRNA IN RYE SEEDLINGS

In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.     

FIBER-OPTIC PLANT TISSUES: SPECTRAL DEPENDENCE IN DARK-GROWN AND GREEN TISSUES*

Abstract— The fiber-optic properties of etiolated plant tissues can be used to detect and characterize pigment absorption in vivo. Transmission spectra of light guided through several monocot and dicot etiolated tissues show a decreasing red/far red ratio with increasing tissue length. Absorption bands attributable both to vacuolar pigments such as anthocyanins and to chloroplast pigments lead to the conclusion that the guided light passes through both vacuole and cytoplasm. As etiolated tissue becomes green under white light treatment, the red/far red ratio also changes, the nature of the change depending upon the tissue involved. The blue/red ratio also changes both with increasing length of etiolated tissue and during the greening process, with the changes again dependent on the tissue involved. The spectral dependence of the light-guiding phenomenon in dark grown and green plants may have implications for physiological responses mediated by phytochrome.     

THE EFFECT OF CYTOKININS ON GROWTH and PIGMENT ACCUMULATION OF RADISH SEEDLINGS (RAPHANUS SATIVUS L.) GROWN IN THE DARK and AT DIFFERENT LIGHT QUANTA FLUENCE RATES*

Abstract— The dependency of cytokinin effects upon irradiance was studied with radish seedlings ( Raphanus sativus L. cv. Saxa Treib). Kinetin (6-furfurylamino-purine) or BAP (6-benzylamino-purine) were applied via the roots of plants growing either in continuous darkness or under high (90 Wm^-2) or low intensity white light (10Wm^-2). Apart from the different development of plants at low and high fluence rates, the following cytokinin effects were found:
(1) Both cytokinins acted in a similar manner on growth characteristics and pigment accumulation at high and low light conditions, BAP being in many cases more effective than kinetin.
(2) When compared with the control, the cytokinins suppressed hypocotyl and root lengthening in the dark and light-grown plants. In darkness they led to increased cotyledon areas, whereas in the light the leaf expansion was suppressed.
(3) In the etiolated and low light grown plants, the anthocyanin content of the hypocotyls was enhanced due to the action of cytokinins, whereas under high light the anthocyanin accumulation was decreased.
(4) In the cotyledons of etiolated plants, more phototransformable protochlorophyll(ide) and more carotenoids were formed when cytokinins were present. In green leaves the carotenoid content was diminished due to the action of cytokinins, particularly in plants grown in strong light. The chlorophyll a/b ratio was increased in the cytokinin-treated plants in most cases.
The results suggest a light dependency of the cytokinin effects. It is believed that the response of a plant towards exogenously applied cytokinins is similar to that with high intensity light.     

THE EFFECT OF RED AND FAR RED LIGHT ON CAROTENOID AND CHLOROPHYLL FORMATION IN PEA SEEDLINGS

Abstract— Treatment of etiolated pea seedlings with a short exposure to red light caused a stimulation of growth (size and dry wt production) and carotenoid synthesis during the following 48 hr compared with seedlings kept entirely in darkness.The effect is nullified by a following dose of far red light and thus the phenomenon is probably phytochrome-controlled.
Similar treatment with red light one hour before continuous illumination with white light tended to reduce the lag period for chlorophyll synthesis.Again a following dose of far red light reversed this response.     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill-activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.     

In vivo PHYTOCHROME-MEDIATED PERCEPTION OF REFLECTED LIGHT SIGNALS

The spectrophotometric assay of phytochrome in vivo in etiolated plant material was used to determine the effects of changes in reflected light on the state of the photoreceptor in etiolated seedlings exposed simultaneously to direct and reflected light. Changes in reflected light that were small in terms of the total (direct + reflected) radiation incident on the seedlings produced detectable changes in the state of phytochrome in vivo. The contribution of reflected light to the state of phytochrome in vertical organs was greater than expected from its low contribution to total incident light. These data from laboratory studies complement and are consistent with results of field studies on the effects of light reflected from neighboring vegetation on plant growth under natural radiation conditions.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

PHOTORESPONSES OF Arabidopsis SEEDLINGS EXPRESSING AN INTRODUCED OAT phyA cDNA: PERSISTENCE OF ETIOLATED PLANT TYPE RESPONSES IN LIGHT-GROWN PLANTS

The photocontrol of hypocotyl elongation has been studied in etiolated and light-grown wild type (WT) Arabidopsis thaliana (L. Heynh) seedlings, and in two homozygous isogenic lines that have been transformed with the oat phy A gene coding sequence under the control of the cauliflower mosaic virus (CaMV) 35S promoter. For etiolated seedlings the inhibition of hypocotyl elongation by continuous broad band far-red light (FR) is saturated at much lower photon fluence rates in the transgenic seedlings compared with WT seedlings. Furthermore, whereas de-etiolation of WT seedlings leads to loss of responsiveness of the hypocotyls to prolonged FR, de-etiolated transgenic seedlings continue to show a pronounced FR-mediated inhibition of elongation. This may reflect the persistence of a FR-high irradiance response (HIR) mediated by the introduced oat phytochrome A. Although the hypocotyls of light-grown transgenic seedlings display a qualitatively normal end-of-day FR growth promotion, such seedlings display an aberrant shade-avoidance response to reduced red:far-red ratio (R:FR). These results are discussed in relation to the proposal that the constitutive expression of phytochrome A leads to the persistence of photoresponse modes normally restricted to etiolated plants.     

PHYTOCHROME-MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN PARTLY DE-ETIOLATED MUSTARD (Sinapis alba L.) SEEDLINGS

Abstract— Hypocotyl elongation in etiolated mustard ( Sinapis alba L.) seedlings is known to be controlled by phytochrome (Pfr) through a threshold mechanism. The Pfr threshold value required to suppress hypocotyl growth was low (3 times 10⁻²% Pfr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). In the present study the question was addressed whether the threshold control by Pfr of hypocotyl elongation also operates in light-pretreated, partly de-etiolated seedlings after transfer to darkness. The experimental results show that this is the case. Calculation of the threshold level in far-red light pretreated seedlings led to a very low value (3 times 10⁻⁷%) compared to etiolated seedlings (3 times 10⁻²%). In red light pretreated seedlings the threshold level was calculated to be 9 times 10⁻⁷%. Since the light pretreatment affected the rate of degradation of phytochrome strongly (half-life of Ptot in continuous red light was found to be 35 min in far-red pretreated instead of 47 min in etiolated material), the difference in threshold level between far-red and red pretreated material cannot be interpreted unambiguously. However, the conclusion can be drawn that light nretreatment strongly increases the degradation rate of Pfr and decreases the threshold level.     

CIRCANNUAL COURSE OF PHOTOMORPHOGENETIC REACTIVITY IN ETIOLATED BEAN SEEDLINGS

Hook opening and leaf expansion, measured 24 h after standard red light illumination, were considered as parameters determining the photoactivity of etiolated Phaseolus vulgaris L. seedlings. The experiment was repeated every h during one 17-h period each month for 1 year. The mean values for each experimental period indicated that the photoactivity of the etiolated seedlings changed markedly throughout the year according to a seasonal pattern.     

CHLOROPLAST PIGMENTS and THEIR BIOSYNTHESIS IN RELATION TO LIGHT INTENSITY*

Abstract— Depending on the light intensity that they received during growth, radish seedlings altered not only the pigment and quinone composition of the thylakoid membrane but also the chloroplast ultrastructure. In strong light, sun chloroplasts of radish were very similar to those from sun leaves of beech trees, while those developed under under dim light possessed a typical shade chloroplast. Radish shade chloroplasts contained a higher chlorophyll content and a higher concentration of xanthophylls resulting in a lower xanthophyll to carotene ratio as compared to sun chloroplasts. Chloroplasts from radish grown in strong light showed a much higher activity in their terpenoid metabolism than plastids from shade plants. Chlorophylls and carotenoids which are involved in the absorption of light and the transfer of energy during photosynthesis were labeled by [³H]-mevalonate to a much higher degree in plastids from sun leaves as compared to plastids from shade leaves. This shows that in strong light where pigments are continuously broken down and resynthesized in order to maintain photosynthesis, chlorophylls and carotenoids exhibit a much higher turnover rate than the pigments of shade plants.     

Comparative photobiology of growth responses to two UV-B wavebands and UV-C in dim-red-light- and white-light-grown cucumber (Cucumis sativus) seedlings: physiological evidence for photoreactivation

We examined the influence of short-term exposures of different UV wavebands on the elongation and phototropic curvature of hypocotyls of cucumbers (Cucumis sativus L.) grown in white light (WL) and dim red light (DRL). We evaluated (1) whether different wavebands within the ultraviolet B (UV-B) region elicit different responses; (2) the hypocotyl elongation response elicited by ultraviolet C (UV-C); (3) whether irradiation with blue light-enriched white light (B/WL) given simultaneous with UV-B treatments reversed the effect of UV in a manner indicative of photoreactivation; and (4) whether responses in WL-grown plants were similar to those grown in DRL. Responses to brief (1-100 min) irradiations with three different UV wavebands all induced inhibition of elongation measured after 24 h. When WL-grown seedlings were irradiated with light containing proportionally greater short wavelength UV-B (37% of UV-B between 280 and 300 nm), inhibition of hypocotyl elongation was induced at a threshold of 0.5 kJ m(-2), whereas exposure to UV-B including only wavelengths longer than 290 nm (and only 8% of UV-B between 290 and 300 nm) induced inhibition of hypocotyl elongation at a threshold of 1.6 kJ m(-2). The UV-C treatment induced reduction in elongation at a threshold of <0.01 kJ m(-2) for DRL-grown plants and <0.03 kJ m(-2) for WL-grown plants. B/WL caused 50% reversal of the short-wavelength UV-B-induced inhibition of elongation in DRL-grown seedlings but did not reverse the effect of long-wavelength UV-B. B/WL caused 30% reversal of the UV-C-induced inhibition of elongation in WL-grown seedlings but did not affect the response to short-wavelength UV-B. Short-wavelength UV-B also induced positive phototropic curvature in both types of seedlings, and this was reversed 60% or completely in DRL-grown and WL-grown seedlings, respectively. The similarity of responses between the etiolated (DRL-grown) and de-etiolated (WL-grown) seedlings indicates that the short-wavelength specific response may be relevant to natural light environments, and the apparent photoreactivation implicates DNA damage as the sensory mechanism for the response.     

ON THE POLARITY OF GREENING IN PRIMARY LEAVES OF DARK AND LIGHT-GROWN SEEDLINGS OF Triticum aestivum*

Abstract— In primary leaves of four-day-old dark-grown wheat seedlings the capability to accumulate chlorophyll under white light is highest in the tissues of the apical region. It declines though, as the dark-grown seedling ages and the primary leaf grows longer. This decrease is lower in the basal than in the apical regions of a leaf, which results in an inverted polarity of the capability to accumulate chlorophyll If the seedlings are grown under continuous white light applied from four different directions, the high capability of greening observed in the apical tissues of young leaves stays at that level even in old seedlings: the polarity does not change during culturing. The saturation-level of greening is much higher in the tissues taken from the apical region of a primary leaf than in those from the basal region.     

THE EFFECT OF NORFLURAZON ON PHOTOTROPIC REACTION IN Phaseolus aureus Roxb. SEEDLINGS

Abstract. The phototropic response of norflurazon-treated mung bean seedlings has been studied to evaluate the possible role of carotenoids, carotenoid-derived growth substances, or other factors in the perception/reaction system. Phototropism was slowed significantly in plants grown in white and blue light in the presence of norflurazon. This effect was evident in norflurazon-bleached seedlings, as well as in those whose pigment system was not affected, due to a shorter period of herbicide action. The possible modes of norflurazon action are discussed.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION AND BINDING IN DICOTYLEDONOUS vs MONOCOTYLE-DONOUS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— The irradiance and wavelength dependence of phytochrome destruction in vivo was analysed in etiolated cotyledons of Cucurbita pepo L. and etiolated seedlings of Amaranthus caudatus L. In contrast to grass seedlings, the rate of P _tot destruction could only be saturated by light sources that establish relatively high P _fr levels (about 50% of total phytochrome, corresponding to the photostationary state established by 693 nm light). To explain the irradiance dependence of P _tot destruction in dicots at irradiances above 0.1 Wm^-2, where the light reaction is at least one order of magnitude faster than P _fr destruction, we suggest there is a fast intercalary dark reaction between photoreaction and destruction. This dark reaction is probably—as in grass seedlings—the binding of P _fr to a receptor site. We conclude that the differences between dicots and grass seedlings with respect to the phytochrome system are of a quantitative rather than a qualitative nature.     

Phytochrome-regulated expression of genes encoding light-harvesting chlorophyll a/b-protein in two long hypocotyl mutants and wild type plants of Arabidopsis thaliana

The cab genes which encode the light-harvesting chlorophyll a/b-protein (LHCP) are expressed normally with respect to phytochrome regulation in the hy-3 and hy-5 long hypocotyl mutants of Arabidopsis thaliana. In etiolated seedlings of these mutants as well as of the wild type, 1 min of red light elevates cab mRNA levels substantially within 2 h; this increase is reversed if far-red light is given immediately after the red light treatment. We conclude that the genetic defects in these mutants do not affect steps in the signal transduction pathway leading to the regulated expression of cab genes. Additionally, the mRNA from one of the three known A. thaliana cab genes, AB140, is similar in quantity to the mRNAs from the other two, AB165 and AB180, in dark-grown seedlings of hy-3 and hy-5 as well as the parent A. thaliana (Landsberg) after a brief red light treatment. This aspect of cab gene expression differs from the strain Columbia of A. thaliana in which AB140 mRNA is the predominant message. In mature white light-grown plants of the strain Columbia, AB140 as well as a combination of AB165 and AB180 mRNAs are expressed at high levels, suggesting that AB165 and/or AB180 may be developmentally regulated.     

SOME FACTORS AFFECTING THE THERMOCHEMILUMINESCENCE OF CHLOROPHYLL

Abstract— The therrnochemiluminescence of the chloroplasts from normal lettuce seedlings is enhanced by the addition of β carotene but not by chlorophyll a .The thermochemiluminescence of chloroplasts from etiolated seedlings is enhanced by neither β carotene nor chlorophyll a .A variety of electron donors were substituted for β carotene in the mixtures with chlorophyll a .The most active were squalene, β carotene, vitamin A, and nerolidol.
The isoprene structure (-CH₂-CCH₃=CH-CH₂-) appears to be a necessary but not sufficient requirement for energy transfer during thermochemiluminescence.Changes in the absorption spectra indicate that both chlorophyll and carotene are destroyed during thermochemiluminescence.     

RESPONSE OF LIGHT-GROWN WILD-TYPE and LONG HYPOCOTYL MUTANT CUCUMBER PLANTS TO END-OF-DAY FAR-RED LIGHT

Abstract— A long-hypocotyl mutant ( lh ) of cucumber ( Cucumis sativus L.) has been studied which has previously been shown to lack phytochrome control of growth in de-etiolated seedlings and thought to be modified with respect to the light-stable type of phytochrome. We have analyzed the response of lh mutant and isogenic wild-type (WT) plants to daily treatment with end-of-day far-red light (EODFR). Only the WT responded to this treatment resulting in a large increase in internode length; an increase in petiole length; changes in leaf development (increased area, decreased thickness and reduction in indentation); redistribution of dry matter from leaf blades to stem; increased apical dominance and promotion of tendril formation. There were only small or no significant effects on chlorophyll and total carotenoid content, chlorophyll alb ratio, soluble protein levels and net photosyn-thetic rates. The lh mutant failed to respond to EODFR treatment, and had the appearance of a shade-avoiding plant growing in extreme shade. The lh mutant appears to completely lack the phytochrome responses attributable to the type of phytochrome that is active in shade detection. A discussion of the possible roles of the stable and labile types of phytochrome in light grown plants follows.