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The present paper is part of a research program which aims at a quantitative analysis of the effects of light and gibberellic acid (GA) on growth of the first foliage leaf in durum wheat ( Triticum durum Desf.). Since leaf growth is the combined result of the increase in cell number (cell division) and cell enlargement, the influence of light and GA treatment on cell division in the basal meristem of the first leaf in two cultivars, Cappelli and Creso, was investigated. Creso is a short-strawed cultivar carrying the Gai 1 gene which influences both plant height and insensitivity to applied GA. Cell division, as measured by mitotic index, was similar in darkness, continuous red light and dichromatic irradiation (far-red plus red), while lower mitotic rates were observed under continuous far-red light: this indicates that the response of cell division is modulated by a high-irradiance reaction of phytochrome in both cultivars. The two cultivars showed different responses to blue light. In Cappelli, blue light and dichromatic irradiation (blue plus red) gave lower mitotic indices than the dark control, indicating the action of a specific blue-light-absorbing photoreceptor, whereas in Creso the response kinetics to all light regimes which included blue light were more complex. On the basis also of the results obtained with GA application in Cappelli, it appears that (i) the hormonal treatment is able to change the pattern of mitotic index only in the presence of the action of a blue-light receptor and (ii) the different responses of the two cultivars could be the result of different endogenous hormonal levels. The importance of the observations in relation to the data for first-leaf longitudinal growth reported in a previous paper (Baroncelli et al. 1984, Planta 160, 298-304) is discussed. [ABSTRACT FROM AUTHOR]

In photosynthetic cells the plastidic ascorbate-glutathione pathway is considered the major sequence involved in the elimination of active oxygen species. Ascorbate peroxidase (APO; EC 1.11.1.11) is an essential constituent of this pathway. In the present paper control of the appearance of APO was studied in the cotyledons of mustard ( Sinapis alba L.) seedlings with the following results: (i) Two isoforms of APO (APO I, APO II) could be separated by anion-exchange chromatography; APO I is a plastidic protein, while APO II is extraplastidic, very probably cytosolic. (ii) The appearance of APO is regulated by light via phytochrome. This control is observed with both isoforms. Moreover, a strong positive control over APO II appearance (very probably over APO II synthesis) is exerted by photooxidative treatment of the plastids. (iii) Additional synthesis of extraplastidic APO II is induced by a signal created by intraplastidic pigment-photosensitized oxidative stress. The response is obligatorily oxygen-dependent and abolished by quenchers of singlet oxygen such as α-tocopherol and p-benzoquinone. (iv) A short-term (4 h) photooxidative treatment suffices to saturate the signal. Signal transduction cannot be abolished or diminished by replacing the plants in non-photooxidizing conditions. Several observations indicate that control of APO synthesis by active oxygen is not an experimental artifact but a natural phenomenon. [ABSTRACT FROM AUTHOR]

In a preceding paper (Oelmüller and Mohr 1986, Planta 167, 106-113) it was shown that in the cotyledons of the mustard ( Sinapis alba L.) seedling the integrity of the plastid is a necessary prerequisite for phytochrome-controlled appearance of translatable mRNA for the nuclear-encoded small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP). It was concluded that a signal from the plastid is essential for the expression of nuclear genes involved in plastidogenesis. The present study was undertaken to characterize this postulated signal. Chloramphenicol, an inhibitor of intraplastidic protein synthesis and Norflurazon, an inhibitor of carotenoid synthesis (to bring about photooxidative sensitivity of the plastids) were applied. We obtained the following major results. (i) After a brief period of photooxidative damage a rapid decrease of the above translatable mRNAs was observed. Conclusion: the signal is short-lived and thus required continually. (ii) Once the plastids became damaged by photooxidation, no recovery with regard to nuclear gene expression was observed after a transfer to non-damaging light conditions. Conclusion: even a brief period of damage suffices to prevent production of the signal. (iii) Chloramphenicol inhibited nuclear gene expression (SSU, LHCP) and plastidic development when applied during the early stages of plastidogenesis. Once a certain stage had been reached (between 36-48 h after sowing at 25° C) nuclear gene expression became remarkably insensitive toward inhibition of intraplastidic translation. Conclusion: a certain developmental stage of the plastid must be reached before the signal is released by the plastid. (iv) Under the growth conditions we adopted in our experiments the plastids in the mesophyll cells of mustard cotyledons developed essentially between 36 and 120 (-144) h after sowing. Only during this period could translatable mRNAs for SSU and LHCP be detected. Conclusion: the signal is released by the plastids only during this time span. [ABSTRACT FROM AUTHOR]

Dry lettuce ( Lactuca sativa L.) 'seeds' (achenes) contain α-galactosidase (EC 3.2.122) at a level which is maintained in the imbibed dormant state in darkness. Both red light (R) and gibberellic acid promote an increase in enzyme activity several hours prior to the completion of germination. Germination and enzyme activity are not essentially linked, however, for the latter can increase while the former is inhibited. α-Galactosidase activity increases within the cotyledons and the endosperm following R stimulation, but the axis is essential to perceive the stimulus and to promote and maintain the increase in enzyme activity. A diffusible factor (or factors) is produced by and-or released from irradiated axes, and it migrates to the cotyledons (and possibly endosperm) to promote the increase in α-galactosidase activity. Gibberellic acid, particularly in the presence of benzyladenine, can replace the requirement for irradiated axes. [ABSTRACT FROM AUTHOR]

Seeds of Bromus sterilis L. germinated between 80-100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response of B. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination. [ABSTRACT FROM AUTHOR]

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, the far-red light absorbing form of phytochrome (e.g., by [P]≈0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P, 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. 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 destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P established at the onset of darkness rather than by the actual P level present during the growth period. [ABSTRACT FROM AUTHOR]

Fluence rate-response curves were determined for the inhibition of hypocotyl growth in 54 h old dark-grown Sinapis alba L. seedlings by continuous or hourly 5 min red light irradiation (24 h). In both cases a fluence rate-dependence was observed. More than 90% of the continuous light effect could be substituted for by hourly light pulses if the total fluence of the two different light regimes was the same. Measurements of the far red absorbing form of phytochrome ([ P]) and [ P]/[ P tot] (total phytochrome) showed a strong fluence rate-dependence under continuous and pulsed light which partially paralleled the fluence rate-response curves for the inhibition of the hypocotyl growth. [ABSTRACT FROM AUTHOR]

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings ( Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation. [ABSTRACT FROM AUTHOR]

The far-red absorbing form of phytochrome, P, rapidly increases the rate of transfer of β-fructosidase (E.C.3.2.1.26) from the cytoplasm to the cell wall in radish hypocotyls. Far-red light increases the level of enzyme in a particulate fraction: after two hours of light treatment, the particulate enzyme is associated almost exclusively with the endoplasmic reticulum. Transfer from the endoplasmic reticulum to the cell wall involves an incorporation into Golgi bodies and the plasmalemma: these membrane fractions were separated by centrifugation on a discontinuous sucrose density gradient and their degree of purity was determined by the use of known biochemical markers. With respect to β-fructosidase, light controls, via P: (1) the total amount, (2) the incorporation into the endoplasmic reticulum and (3) the transfer to the cell-wall. These three processes have different sensitivities to cycloheximide. [ABSTRACT FROM AUTHOR]

The concept (Burg, 1973) that ethylene mediates the action of phytochrome in seedling photomorphogenesis was tested in the intact mustard ( Sinapis alba L.) seedling. The effect of exogenous ethylene (100 μl l) on five distinct, phytochrome-mediated photoresponses of the cotyledons was investigated. It was found that anthocyanin contents (see Bühler et al., 1978) and phenylalanine ammonia-lyase levels (EC 4.3.1.5) are strongly reduced by ethylene while the capacity of chlorophyll synthesis is considerably enhanced. Levels of glutathione reductase (EC 1.6.4.2) and pools of photoconvertible protochlorophyll(ide) are unaffected by ethylene. It is concluded that these findings are incompatible with the idea that ethylene plays the role of a mediator in phytochrome-induced photomorphogenesis. [ABSTRACT FROM AUTHOR]

A short period (15-30 min) at 30° C promotes germination of seeds of Lactuca sativa L. cv. Repolhuda in darkness. Far-red light reverses this stimulation, and the escape curves for phytochrome and high-temperature action are quite similar, indicating that the two factors act at a common point in the chain of events leading to germination. It is suggested that high temperature acts by decreasing the threshold of the active, far-red absorbing, form of phytochrome (Pfr) needed to promote germination. [ABSTRACT FROM AUTHOR]

While there is a great deal of knowledge regarding plant growth and development in microgravity aboard orbiting spacecraft, there is little information available about these parameters in reduced or fractional gravity conditions (less than the nominal 1 g on Earth). Thus, in these experiments using the European Modular Cultivation System on the International Space Station, we studied the interaction between phototropism and gravitropism in the WT and mutants of phytochrome A and B of Arabidopis thaliana. Fractional gravity and the 1 g control were provided by centrifuges in the spaceflight hardware, and unidirectional red and blue illumination followed a white light growth period in the time line of the space experiments. The existence of red-light-based positive phototropism in hypocotyls of seedlings that is mediated by phytochrome was confirmed in these microgravity experiments. Fractional gravity studies showed an attenuation of red-light-based phototropism in both roots and hypocotyls of seedlings occurring due to gravitational accelerations ranging from 0.l to 0.3 g. In contrast, blue-light negative phototropism in roots, which was enhanced in microgravity compared with the 1 g control, showed a significant attenuation at 0.3 g. In addition, our studies suggest that the well-known red-light enhancement of blue-light-induced phototropism in hypocotyls is likely due to an indirect effect by the attenuation of gravitropism. However, red-light enhancement of root blue-light-based phototropism may occur via a more direct effect on the phototropism system itself, most likely through the phytochrome photoreceptors. To our knowledge, these experiments represent the first to examine the behavior of flowering plants in fractional or reduced gravity conditions. [ABSTRACT FROM AUTHOR]

Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato ( Solanum lycopersicum L.) that the apical hook curvature is exaggerated by light. Experiments with several tomato cultivars and phytochrome mutants, irradiated with red and far-red light either as a brief pulse (Rp, FRp) or continuously (Rc, FRc), revealed: the hook-exaggeration response is maximal at the emergence of the hypocotyl from the seed; the effect of Rp is FRp-reversible; fluence–response curves to a single Rp or FRp show an involvement of low and very low fluence responses (LFR, VLFR); the effect of Rc is fluence-rate dependent, but that of FRc is not; the phyA mutant ( phyA hp-1) failed to respond to an Rp of less than 10−2 μmol m−2 and to an FRp of all fluences tested as well as to FRc, thus indicating that the hook-exaggeration response involves phyA-mediated VLFR. The Rp fluence–response curve with the same mutant also confirmed the presence of an LFR mediated by phytochrome(s) other than phyA, although the phyB1 mutant ( phyB1 hp-1) still showed full response probably due to other redundant phytochrome species (e.g., phyB2). Simulation experiments led to the possible significance of hook exaggeration in the field that the photoresponse may facilitate the release of seed coat when seeds germinate at some range of depth in soil. It was also observed that seed coat and/or endosperm are essential to the hook exaggeration. [ABSTRACT FROM AUTHOR]

The mossPhyscomitrella patensis so far the only plant species in which it is possible for nuclear genes to be modified by homologous recombination at a reasonably efficiency. Here we describe the use of homologous recombination for another moss,Ceratodon purpureus. Our approach is based on the repair of theptr116mutant allele. In this mutant, codon 31 of the heme oxygenase geneCpHO1is mutated to a stop codon. Heme oxygenase is necessary for the conversion of heme to biliverdin, the precursor of the phytochrome chromophore. Thus, inptr116the phytochrome-mediated responses of phototropism, chlorophyll accumulation and branching are lost. Protoplast transformation with DNA encoding the wild-type protein resulted in a rescue of 0.8% of regenerated protoplasts. In about half of the analyzed lines, formation ofCpHO1concatemers was observed at theCpHO1locus, whereas in the other half, the mutantCpHO1gene was replaced by a single DNA copy. This gene repair led to the exchange of single bases, and thus provides the first demonstration of efficient site-directed mutagenesis in a plant nuclear genome. Our studies also revealed an effective mechanism for gene inactivation inCeratodon. When wild-type protoplasts were transformed with intact or modifiedCpHO1genes, approximately 40% of regenerated protoplasts showed theptrphenotype. [ABSTRACT FROM AUTHOR]

The expression of the gene coding for the carotenogenic enzyme phytoene synthase is highly regulated. To study this, its promoter and truncated versions thereof were translationally fused to the luciferase gene as a reporter and these constructs were used to transform Arabidopsis thaliana. The full-length promoter was shown to be active in the dark, but mediated positive responses towards different light qualities (far-red, red, blue and white light). Among the herbicides tested, norflurazon and gabaculine showed no notable effects, while CPTA abolished light induction completely. Response towards different light qualities was mediated by a TATA box-proximal promoter region up to position –300, containing G-box-like elements involved in the distinction of different monochromatic light qualities applied. This is detected in electrophoretic mobility shift assays (EMSAs), which reveal differential complex formation. A TATA box distal region of the promoter was shown to be responsible for a high basal promoter activity that was not modulated by different light qualities. Using EMSAs, a novel cis-acting element ATCTA occurring in tandem between positions –854 and –841 proved to be decisive in this respect. The motif was found in several other promoter regions involved in carotenoid and tocopherol biosynthesis, as well as in the promoter regions mediating the expression of photosynthesis-related genes. The functional equivalence of the motifs was shown by successfully using the respective regions in EMSAs. We conclude that the ATCTA motif represents an element capable of mediating a coordinated regulation of these pathways at the transcriptional level. [ABSTRACT FROM AUTHOR]

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one. [ABSTRACT FROM AUTHOR]

A single pulse of red light (R) given to 4-d-old etiolated high-pigment-1 ( hp-1) mutant tomato ( Solanum lycopersicum L.) seedlings followed by a 3-d dark period is demonstrated to result in a block of greening in subsequent white light. Wild-type seedlings green normally under this regime. The block of greening in the hp-1 mutant depends on the length of the dark period before and after the R pulse and operates via the low-fluence-response mode of phytochrome action. This block of greening takes place in hp-1 double mutants lacking either phytochrome A or phytochrome B1, but is absent in the hp-1 triple mutant lacking both phytochromes A and B1. These observations enable a screen to be devised for new phytochrome B1 mutants either within the photoreceptor or mutants defective in phytochrome B1-signalling steps which result in loss of capacity to green, by mutagenising the phytochrome A-deficient hp-1, fri double mutant. [ABSTRACT FROM AUTHOR]

In darkness, protonemal filaments of Ceratodon purpureus (Brid.) grow negatively gravitropically (upwards). Red light induces a positive phototropic response mediated by the photoreceptor phytochrome. A red light treatment also has an inhibitory effect on the gravitropic response, an effect also mediated by phytochrome. In this study the effects of blue light on phototropism and on gravitropism were analysed. Unilateral blue light resulted in only a weak phototropic response, but markedly randomised growth direction. Blue light given together with a gravitropic stimulus reversed the gravitropism, changing it from negative to positive (filaments grow downward). The effect of blue light was also analysed with the mutant ptr116, which is defective in the biosynthesis of the phytochrome chromophore, and in a newly isolated mutant wwr2, which is positively gravitropic in darkness. Blue light induced the same reversal of gravitropism in ptr116 as in the wild type, indicating that phytochrome is not involved in this process. In wwr2 the direction of gravitropism was unaltered by the blue light treatment. Light also affects chlorophyll content and the size of plastids, potential statoliths for gravitropism. Red light induced an increase in plastid size and chlorophyll content in the wild type but not in ptr116. Blue light induced a similar change in wild type plastids. It seems as though light-induced alterations of gravitropism are not simply mediated by alterations in plastid properties, and that red light and blue light evoke fundamentally different responses. [ABSTRACT FROM AUTHOR]

Unifoliated plants of Lolium temulentum L. Ceres were induced to flower by a unique 24-h long day (LD) consisting of the extension of the regular 8-h short day (SD) (400 μmol photons·m·s, fluorescence + incandescence) with incandescence at 10-15 μmol photonsm ·s. The polyadenylated-RNA complement of leaf blade tissues was analysed at 4-h intervals during the photoperiod extension in LD vs. SD, by using two-dimensional polyacrylamide gel electrophoresis to resolve in-vitro-translated products. Of the 991 spots that were analysed, none appeared or disappeared during the inductive cycle, i.e. no qualitative effect of floral induction was detected, at any time. Sixty-eight spots were found whose intensity was influenced by lengthening of the photoperiod; 50 of them, i.e. ca. 5% of the population analysed, were affected before the end of the extension period and were thus potentially related to floral induction. Many of these RNAs were not quantitatively constant during a 24-h cycle in SD. Seven of them oscillated according to the 'light-on' and the 'light-off' signals, among which three seemed to be controlled by phytochrome since their relative amount increased under the standard light conditions but decreased under incandescence even faster than in darkness. The large majority of other RNAs varied with a timing that was not clearly driven by the alternation of light and darkness, indicating that genes related to the biological clock may be especially sensitive to the lengthening of the photoperiod. Furthermore, seven spots were observed that underwent a phase-shift in LD, which consisted, for six of them, of a phase advance of 4-8 h. The steady-state level of CAB mRNA was analysed because the CAB gene family (encoding the chlorophyll a/b-binding proteins of the light-harvesting complexes) is known to be controlled both by the biological clock and phytochrome. In SD, the level was high in the light and low in darkness; the fluctuation was conducted by a circadian rhythm. When plants were exposed to the inductive LD, the peak of mRNA accumulation that was expected according to the endogenous rhythmicity was abolished, possibly because of the change in light quality during the LD extension. [ABSTRACT FROM AUTHOR]

When prothalli of Adiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389-398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 μm·min between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement. [ABSTRACT FROM AUTHOR]

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed. [ABSTRACT FROM AUTHOR]

Etiolated seedlings of tobacco ( Nicotiana tabacum L.) were exposed to single light pulses predicted to establish different proportions of phytochrome in its far-red absorbing form (Pfr/P). The angle between the cotyledons was compared in wild-type and transgenic seedling overexpressing Avena phytochrome A over the range of both very low-fluence responses (VLFR) and low-fluence responses (LFR). The unfolding of the cotyledons increased linearly for 24 h after the light pulse. At this time the Pfr/P-response curve showed two linear segments. The segment below a calculated Pfr/P = 3% (i.e. VLFR) was steeper than the segment above 3% (i.e. LFR). In the VLFR range the slope was almost threefold higher in transgenic than wild-type seedlings. However, in the LFR range the difference was less than 50%. From these data we propose that Avena phytochrome A makes a higher contribution to VLFR than LFR in etiolated tobacco seedlings. [ABSTRACT FROM AUTHOR]

Nitrite reductase (NIR, EC 1.7.7.1) - a key enzyme of nitrate assimilation - is known to be induced by nitrate and light. In the present work with barley ( Hordeum vulgare L.) seedlings, control of NIR gene expression by light and its dependency on intact plastids was studied. The major results were as follows: (i) It was confirmed that a single isoform of NIR occurs in shoot and root. (ii) Any light-mediated increase of NIR activity is strictly correlated with an increase in cross-reacting material. (iii) Exogenous nitrate is absolutely required for NIR synthesis and the appearance of NIR transcripts. The nitrate effect is saturated at 10 mM. (iv) Light-pulse experiments and long-term light treatments indicate the involvement of phytochrome in the light response. (v) While a small light effect possibly occurs in the root, a strong action of phytochrome was observed in the shoot. (vi) Photooxidative treatments of plastids led to a strong decrease of the NIR level and almost abolished the appearance of NIR transcripts. (vii) With plastids intact, application of nitrate (10 mM) caused the appearance of a considerable transcript level even in darkness. Light treatments in the presence of nitrate stimulated the transcript level further. (viii) Since a correlation between transcript level and rate of NIR increase was found it was concluded that in barley - in contrast to mustard, spinach and tobacco - NIR gene expression is controlled predominantly, if not exclusively, at the level of transcript accumulation. [ABSTRACT FROM AUTHOR]

Fluence rate-response curves were generated for red-, far-red-, and blue-light-stimulated apical-hook opening in seedlings of several photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. Compared to wild-type plants, hook opening was reduced in the phytochrome-deficient hy1, hy2, and hy6 mutants in red and far-red light at all fluence rates tested, and in low-fluence blue light, but was normal under high-irradiance blue light. In contrast, the blue-light-response mutants ( blu1, blu2, and blu3) lacked the high-irradiance-dependent hook-opening response in blue light while hook opening was normal in low-fluence blue light and in red and farred light at all fluence rates tested. Hook opening in the phytochrome-B-deficient hy3 mutant was similar to wild type in all light conditions tested. The effects of the different mutations on light-induced hook opening indicate that a phytochrome(s) other than phytochrome B mediates hook opening stimulated by red, far-red and lowfluence blue light, while a blue-light-absorbing photoreceptor mediates the blue-light-sensitive high-irradiance response. Although the phytochrome and blue-light photosensory systems appear to work independently for the most part, some of their signal-transduction components may interact since the hy4, and hy5 mutants showed reduced hook-opening responses under conditions dependent on the phytochrome and blue-light-photosensory systems. [ABSTRACT FROM AUTHOR]

Anthocyanin synthesis in the broom sorghum, Sorghum bicolor Moench cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama, is mediated separately or synergistically by an ultraviolet light-B (UV-B) photoreceptor and phytochrome. When seedlings were exposed to moderate low temperatures ranging from 12 to 20° C before irradiation, only the phytochrome-mediated anthocyanin synthesis was markedly enhanced compared with the control, which was kept throughout at 24° C; synthesis mediated by the UV-B photoreceptor was unaffected. The effectiveness of an exposure to 20° C increased as the duration of exposure increased up to 24 h and as the time of exposure became closer to the time of irradiation. However, when seedlings were exposed to 20° C from after irradiation until harvest, anthocyanin syntheses induced by both UV-B and red light were equally suppressed, probably due to the general reduction of metabolism involved in anthocyanin synthesis that is a consequence of lower temperature. The results support the view that the signal transduction of the pyhtochrome system is different from that of the UV-B photoreceptor, and indicate that the phytochrome system may involve a step or steps which are amplified by a previous exposure to the moderate low temperature. [ABSTRACT FROM AUTHOR]

An oat ( Avena sativa L.) plant contains at least three phytochromes, which have monomeric masses of 125, 124, and 123 kilodaltons (kDa) (Wang et al., 1991, Planta 184, 96-104). The 124-kDa phytochrome is most abundant in dark-grown seedlings, while the other two phytochromes predominate in light-grown seedlings. Using three monoclonal antibodies, each specific to one of the three phytochromes, we have monitored by immunoblot assay the expression of these three phytochromes in the 5 d following onset of imbibition of seeds. On a per-organism basis, each of these three phytochromes increased in abundance for the first 3 d in the light, or for the first 4 d in darkness, after which they each began to decrease in quantity. When 3-d-old dark-grown seedlings were transferred to the light, the abundance of each of these three phytochromes decreased both in absolute amount and relative to the phytochrome levels in control seedlings kept in darkness. In contrast, when 3-d-old light-grown seedlings were transferred to darkness, the abundance of the 124-kDa and 125-kDa phytochromes increased while that of 123-kDa phytochrome remained unchanged. In each case, the level of phytochrome was greater than that of control seedlings maintained in the light. Thus, in addition to temporal regulation, all three phytochromes exhibit photoregulated expression at the protein level, although the magnitude of this photoregulation varies substantially. [ABSTRACT FROM AUTHOR]

Photon fluence rate-response curves at different wavelengths were generated for the light-induced inhibition of hypocotyl elongation in seedlings of wildtype and photomorphogenic mutants of Arabidopsis thaliana. (L.) Heynh. Treatment of wild-type seedlings with continuous low-fluence-rate light (< 1.0 μmol photons · m · s) induced some inhibition of hypocotyl elongation at all wavelengths tested, with maximum inhibition in blue light. At higher fluence rates, inhibition reached a maximum of 70-80% in UV-A, blue, and far-red light. Fluence rate-response curves for seedlings of blu1, a blue light-response mutant, showed a specific reduction in their response to blue light, but their response to UV-A, red, and far-red light was similar to that in wild-type seedlings. In contrast, the phytochromedeficient mutant hy6 showed a loss of response to lowfluence-rate light at all wavelengths, as well as to highfluence-rate far-red light. However, hy6 seedlings retained sensitivity to high-fluence-rate blue and UV-A light. The data support the conclusion that blue-lightand phytochrome-dependent photosensory systems regulate hypocotyl elongation independently and in an additive manner. Furthermore, hypocotyl inhibition in wild-type, blul, hy6 and blul-hy6 double mutants was indistinguishable in UV-A light, whereas marked differences were observed at other wavelengths, indicating the involvement of a third photosensory system with an absorption maximum in the UV-A. [ABSTRACT FROM AUTHOR]

The phytochrome-deficient aurea mutant of tomato ( Lycopersicon esculentum (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated aurea seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the aurea mutant. The transcript levels for cab and RbcS (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively) in aurea leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO uptake by leaves of the aurea mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and aurea mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in aurea leaves as a consequence of the reduced CO-fixation capacity of the mutant leaves. [ABSTRACT FROM AUTHOR]

Nitrite reductase (NIR; EC 1.7.7.1) - a key enzyme of nitrate reduction - is known to be induced by nitrate and light. In the present study with tobacco ( Nicotiana tabacum L.) seedlings the dependency of NIR gene expression on nitrate, light and a plastidic factor was investigated to establish the nature of the coaction between these controlling factors. A cDNA clone coding for tobacco plastidic NIR was available as a probe. The major results were as follows: (i) The light effect on the appearance of NIR occurred predominantly through phytochrome. However, a specific blue-light effect was also involved. (ii) There was no effect of light on NIR appearance in the absence of nitrate while light exerted a strong effect when nitrate was provided. (iii) Anion-exchange chromatography revealed only a single form of NIR. While experiments involving plastid photooxidation indicated that this NIR is plastidic, a small residual level could not be eliminated by photooxidation. (iv) Northern blot analysis of NIR-transcript levels indicated that a low transcript level existed in the absence of nitrate and light; however, this level appeared to be increased slightly by light (in the absence of nitrate) and by nitrate (in the absence of light). A high transcript level was detected only when light as well as nitrate were provided. A low level was found when the plastids were damaged by photooxidation. It is concluded that plastidic NIR gene expression in tobacco requires positive control by a plastidic factor. Moreover, a synergistic action of phytochrome and nitrate is required to bring about a high transcript level. As found previously with mustard and spinach seedlings, there is no quantitative relationship between the transcript level and the rate of enzyme synthesis. [ABSTRACT FROM AUTHOR]

The physiological responses of transgenic tobacco ( Nicotiana tabacum L.) plants that express high levels of an introduced oat ( Avena sativa L.) phytochrome ( phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005-1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll a∶b ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R ∶ FR. Interpretation of these data is difficult. The depression of the chlorophyll a∶b ratio by low R ∶ FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles. [ABSTRACT FROM AUTHOR]

It is well established that nitrite reductase (NIR; EC 1.7.7.1) a key enzyme of nitrate reduction - is 'induced' by nitrate and light. In the present study with the spinach ( Spinacia oleracea L.) seedling the dependency of NIR appearance on nitrate, light and a 'plastidic factor' was investigated to establish the nature of the coaction between these controlling factors. A cDNA clone coding for spinach NIR was available as a probe. The major results we have obtained are the following: (i) The light effect on the appearance of NIR activity occurs through phytochrome. No specific bluelight effect is involved, (ii) Immunotitration data indicate that light affects the appearance of NIR by inducing the de-novo synthesis of the NIR protein, (iii) A multiplicative relationship exists between the action of nitrate and light on NIR appearance. This indicates that the actions of light and nitrate are indeed independent of each other but that both factors operate on the same causal sequence, (iv) Anion-exchange chromatography revealed only a single form of NIR in spinach. Experiments involving plastid photooxidation indicate that this NIR is exclusively plastidic. (v) Northern blot analysis of NIR mRNA showed a strong increase of the steady-state level in the presence of nitrate whereas light had no effect; NIR mRNA was almost undetectable when the plastids were damaged by photooxidation. It is concluded that NIR gene expression in spinach requires positive control by a 'plastidic factor'. Moreover, nitrate exerts a coarse control at the mRNA level whereas fine tuning of NIR protein synthesis is post-transcriptional and is exerted by light, operating via phytochrome. [ABSTRACT FROM AUTHOR]

A protein-kinase activity which is co-purified with phytochrome from etiolated oat seedlings was investigated in some detail. Whereas phytochrome was always phosphorylated in solution (together with some contaminating protein bands), radioactive phosphate was not found in the phytochrome band after native gel electrophoresis and incubation of the entire gel with labeled ATP. Since protein kinases are usually autophosphorylated under these conditions, the result shows that the kinase activity does not reside in the phytochrome molecule itself. Radioactivity was exclusively detected in a band with the apparent molecular weight 450 kDa; sodium-dodecyl-sulfate gel electrophoresis revealed an apparent molecular weight of 60 kDa for the phosphorylated subunit. The N-terminal amino-acid sequence A L E SKV P W was determined for this subunit which is a potential candidate for the protein kinase. The optimum conditions (pH, metal ion concentration) and kinetics of the phosphorylation reaction were determined. The presumed connection between proteinkinase activity and the signal chain leading from the far-red-absorbing form of phytochrome to physiological responses still awaits elucidation. [ABSTRACT FROM AUTHOR]

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS. The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor. [ABSTRACT FROM AUTHOR]

The influence of red and far-red irradiation on the transport of H and Rb through the plasmalemma was studied using parenchymal protoplasts isolated from Vicia faba leaves. The results indicate that red light stimulates H secretion and the uptake of Rb. Moreover, it has been demonstrated that far-red irradiation acts antagonistically with respect to red light in both these processes. [ABSTRACT FROM AUTHOR]

Internode extension in young, light-grown mustard plants was measured continuously to a high degree of resolution using linear voltage displacement transducers. Plants were grown in background white light (WL) and the first internode was irradiated with supplementary far-red (FR) from fibre-optic light guides, depressing the Pfr/P (ratio of FR-absorbing form of phytochrome to total spectrophotometrically assayable phytochrome) within the internode and causing an acceleration of extension rate. The internode was sensitive to periods of FR as brief as 1 min, with a sharp increase in extension rate occurring after the return to background WL only. The mean latent period of the response to FR was approx. 10 min. Periods of FR longer than approx. 35 min caused an apparently biphasic growth response, with an initial sharp acceleration in extension rate (Phase 1) being followed by a brief deceleration and a further acceleration to a more-or-less steady elevated rate, somewhat less than the first peak (Phase 2). With such longer-term FR, extension rate decelerated upon FR switch-off after a mean lag of approx. 6 min, achieving the prestimulation extension rate within 16 min. The magnitude of the FR-induced increase in extension rate, expressed as a percentage of the rate in WL alone, was an inverse, linear function of the phytochrome photoequilibrium (i.e. Pfr/P, measured in etiolated test material irradiated under the same geometry) over the range 0.17 to 0.63. This relationship was not significantly affected by variations in backround WL fluence rate over the range 50-150 μmol·m·s and was held both for Phase 1 and Phase 2 of the response. The data provide evidence for rapid coupling/uncoupling between phytochrome and its transduction chain in the light-grown plant and for fluence-rate compensation of the regulation of extension rate. The extensive linearity of the relationship between phytochrome photoequilibrium and proportional extension rate increment allows for fine tuning in shade avoidance. The results are discussed with respect to recent evidence on the nature of phytochrome in light-grown plants and in relation to the function of phytochrome in plants growing in the natural environment. [ABSTRACT FROM AUTHOR]

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard ( Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment - operating through phytochrome - prior to nitrate application. This light treatment is almost ineffective - as far as enzyme appearance is concerned - if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h. Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle. [ABSTRACT FROM AUTHOR]

Nitrate-induced and phytochrome-modulated appearance of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of the mustard ( Sinapis alba L.) seedling is strongly affected by externally supplied ammonium (NH). In short-term experiments between 60 and 78 h after sowing it was found that in darkness NH-simultaneously given with NO-strongly inhibits appearance of nitrate-inducible NR and NIR whereas in continuous far-red light-which operates exclusively via phytochrome without significant chlorophyll formation -NH(simultaneously given with NO) strongly stimulates appearance of NR. The NIR levels are not affected. This indicates that NR and NIR levels are regulated differently. In the absence of external NOappearance of NR is induced by NH in darkness as well as in continuous far-red light whereas NIR levels are not affected. On the other hand, in the absence of external NO, exogenous NHstrongly inhibits growth of the mustard seedling in darkness as well as in continuous far-red light. This effect can be abolished by simultaneously supplying NO. The adverse effect of NHon growth ('NH-toxicity') cannot be attributed to pH-changes in the medium since it was shown that neither the growth responses nor the changes of the enzyme levels are related to pH changes in the medium. Non-specific osmotic effects are not involved either. [ABSTRACT FROM AUTHOR]

Phytochrome-controlled appearance of ribulose-1,5-bisphosphate carboxylase (RuBP-Case) and its subunits (large subunit LSU, small subunit SSU) was studied in the cotyledons of the mustard ( Sinapis alba L.) seedling. The main results were as follows: (i) Control of RuBPCase appearance by phytochrome is a modulation of a process which is turned on by an endogenous factor between 30 and 33 h after sowing (25° C). Only 12 h later the process begins to respond to phytochrome. (ii) The rise in the level of RuBP-Case is the consequence of a strictly coordinated synthesis de novo of the subunits. (iii) While the levels of translatable mRNA for SSU are compatible with the rate of SSU synthesis the relatively high LSU mRNA levels are not reflected in the rates of in-vivo LSU or RuBPCase syntheses. (iv) Gene expression is also abolished in the case of nuclear-encoded SSU if intraplastidic translation and concomitant plastidogenesis is inhibited by chloramphenicol, pointing to a 'plastidic factor' as an indispensable prerequisite for expression of the SSU gene(s). (v) Regarding the control mechanism for SSU gene expression, three factors seem to be involved: an endogenous factor which turns on gene expression, phytochrome which modulates gene expression, and the plastidic factor which is an indispensable prerequisite for the appearance of translatable SSU mRNA. [ABSTRACT FROM AUTHOR]

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard ( Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that 'intact plastids' are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some 'plastidic signal' is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle. [ABSTRACT FROM AUTHOR]

Intact mustard seedlings were treated with zeatin and photomorphogenetically active light in different ways: (1) hormone treatment preceding light treatment, (2) light treatment preceding hormone treatment, (3) hormone and light applied simultaneously. Under all experimental conditions the effect of the hormone treatment is multiplicative to the light effect with regard to the increase of cotyledon area. However, the hormone effect is additive to the light effect with regard to increases of the level of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and carotenoid contents. Anthocyanin synthesis is inhibited by exogenous zeatin whereby the concentration response curves are similar, irrespective of the extent of anthocyanin formation mediated by light. However, an interaction was found in the sense that the responsiveness toward zeatin is decreased somewhat by the action of phytochrome. Our results show that the responsiveness to light (via the far-red-absorbing form of phytochrome; P) is not changed by a preceding or simultaneous hormone treatment. Moreover, the responsiveness of the plant to exogenously applied zeatin is not affected - except in anthocyanin synthesis - by a preceding or simultaneous light treatment. We conclude from our results that the action of phytochrome on the developmental processes is not related to cytokinin levels. [ABSTRACT FROM AUTHOR]