Low Fluence Response Research Articles

Phytochrome controls achene germination in Bidens pilosa L. (Asteraceae) by very low fluence response

Achene without ornament of the tegument were light insensitive with germination under all tested light conditions. Achene with verrucose ornament of the tegument presented low germination under darkness and high germination under light conditions. By pre-incubation at 36° C for remotion of pre-existing Pfr and by comparison of results of counting of dark germinating achenes at the end of experiment and daily under dim green safe light (0.001mumol m-2 s-1 nm-1) we concluded that germination was controlled by phytochrome through very low fluence response.

The enhancement of phototropin-induced phototropic curvature in Arabidopsis occurs via a photoreversible phytochrome A-dependent modulation of auxin responsiveness.

The induction of phototropism in etiolated (dark-grown) seedlings exposed to an unidirectional pulse or extended irradiation with low fluence rate blue light (BL) requires the action of the phototropin (nph1) BL receptor. Although cryptochromes and phytochromes are not required for phototropic induction, these photoreceptors do modulate the magnitude of curvature resulting from phototropin activation. Modulatory increases in the magnitude of phototropic curvature have been termed "enhancement." Here, we show that phototropic enhancement is primarily a phytochrome A (phyA)-dependent red/far-red-reversible low fluence response. This phyA-dependent response is genetically separable from the basal phototropin-dependent response, as demonstrated by its retention under extended irradiation conditions in the nph4 mutant background, which normally lacks the basal BL-induced response. It is interesting that the nph4 mutants fail to exhibit the basal phototropin-dependent and phyA-dependent enhancement responses under limiting light conditions. Given that NPH4 encodes a transcriptional activator, auxin response factor 7 (ARF7), we hypothesize that the ultimate target(s) of phyA action during the phototropic enhancement response is a rate-limiting ARF-containing transcriptional complex in which the constituent ARFs can vary in identity or activity depending upon the irradiation condition.

Phytochrome-mediated photoperiod perception, shoot growth, glutamine, calcium, and protein phosphorylation influence the activity of the poplar bark storage protein gene promoter (bspA).

In poplars (Populus), bspA encodes a 32-kD bark storage protein that accumulates in the inner bark of plants exposed to either short-day (SD) photoperiods or elevated levels of nitrogen. In this study, poplars transformed with a chimeric gene consisting of the bspA promoter fused to beta-glucuronidase (uidA) were used to investigate the transcriptional regulation of the bspA promoter. Photoperiodic activation of the bspA promoter was shown to involve perception by phytochrome and likely involves both a low fluence response and a parallel very low fluence response pathway. Activity of the bspA promoter was also influenced by shoot growth. High levels of bspA expression usually occur in the bark of plants during SD but not long day or SD with a night break. When growth was inhibited under growth permissive photoperiods (SD with night break) levels of bark beta-glucuronidase (GUS) activity increased. Stimulating shoot growth in plants treated with SD inhibited SD-induced increases in bark GUS activity. Because changes in photoperiod and growth also alter carbon and nitrogen partitioning, the role of carbon and nitrogen metabolites in modulating the activity of the bspA promoter were investigated by treating excised stems with amino acids or NH4NO3 with or without sucrose. Treatment with either glutamine or NH4NO3 resulted in increased stem GUS activity. The addition of sucrose with either glutamine or NH4NO3 resulted in synergistic induction of GUS, whereas sucrose alone had no effect. Glutamine plus sucrose induction of GUS activity was inhibited by EGTA, okadaic acid, or K-252A. Inhibition by EGTA was partially relieved by the addition of Ca2+. The Ca2+ ionophore, ionomycin, also induced GUS activity in excised shoots. These results indicate that transcriptional activation of bspA is complex. It is likely that SD activation of bspA involves perception by phytochrome coupled to changes in growth. These growth changes may then alter carbon and nitrogen partitioning that somehow signals bspA induction by a yet undefined mechanism that involves carbon and nitrogen metabolites, Ca2+, and protein phosphorylation/dephosphorylation.

Regulation of HEMA1 expression by phytochrome and a plastid signal during de‐etiolation in Arabidopsis thaliana

The synthesis of 5-aminolevulinic acid (ALA) is the rate-limiting step for the formation of all plant tetrapyrroles, including chlorophyll and heme, and regulation of ALA synthesis is therefore critical to plant development. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of this pathway and is encoded by a small family of nuclear HEMA genes. Here, we have used transgenic Arabidopsis (Arabidopsis thaliana L. Col) lines expressing chimeric HEMA1 promoter:gusA fusion genes, combined with RNA gel blot analyses, to characterise the light-mediated regulation of the Arabidopsis HEMA1 gene during de-etiolation. HEMA1 was expressed strongly, but not exclusively, in photosynthetic tissues and was shown to be light regulated at the transcriptional level by the phytochrome family of photoreceptors acting in both the far-red high irradiance and low fluence response modes. The HEMA2 gene, which is expressed only in roots of seedlings, was not light regulated. Analysis of truncated HEMA1 promoter constructs demonstrated that a -199/+252 promoter fragment was sufficient to confer full light-responsiveness to gusA expression. This fragment contained GT-1/I-box and CCA-1 binding sites that are implicated as the light-responsive cis elements. Both the full-length and truncated HEMA1 promoters required the presence of intact chloroplasts for full expression, consistent with previous indications that light and plastid factor signals converge to co-ordinately regulate expression of photosynthesis-related nuclear genes. These results provide the most comprehensive analysis to date of the light-regulation of a tetrapyrrole biosynthetic gene and support a direct link between regulation of HEMA1 transcription and chlorophyll accumulation during seedling de-etiolation.

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis.

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.

New proposal of classification of seeds based on forms of phytochrome instead of photoblastism

The paper proposes the classification of seeds in relation to the forms of phytochrome instead of classical photoblastism. On the basis of published data all seeds have phytochrome and the term photoblastism should be replaced by forms of phytochrome that control germination: 1. Positive photoblastic seeds have phyB (and, to a lesser extent, phyD and phyE) controlling the germination process through Low Fluence Responses (LFR); 2. Negative photoblastic seeds have phyA controlling germination through High Irradiance Responses (HIR) and, when the pre-existing Pfr level is high enough to induce germination in darkness, through LFR by phyB; and 3. light insensitive seeds have phyA controlling germination through Very Low Fluence Responses (VLFR).

Light-induced starch degradation in non-dormant turions of Spirodela polyrhiza.

Continuous red light controls starch degradation in turions of Spirodela polyrhiza [Dölger, K., U. K. Tirlapur and K.-J. Appenroth [1997] Photochem. Photobiol. 66, 126-127 (1997)]. This light could be replaced by repeated red light pulses with the reciprocity law fulfilled over a large range of fluence rates. The effect of red light pulses repeated every 24 or 12 h for 6 days was reversible by subsequent far-red light pulses. In contrast, hourly applied red pulses were irreversible by far-red light. This discrepancy was explained by showing the starch degradation activity of far-red pulses themselves. The investigated process was categorized as a phytochrome low fluence response with an unusual property: requirement of light treatment for several days. A partial fulfillment of this requirement was obtained with a red pulse followed by a dark period and a 24 h continuous irradiation. These results suggest the existence of two separate steps in the process of starch degradation in turions: formation of a sprout (= sink) during the pulse-induced germination, and starch degradation in the storage tissue (= source) induced by the second light treatment.

Photoregulation of asymmetric cell division followed by rhizoid development in the fern Ceratopteris prothalli.

Strap-shaped prothalli of CERATOPTERIS: richardii grown in the dark have an apical meristem, a subapical elongation zone and a basal growth cessation zone [Murata et al. (1997) Plant Cell Physiol. 38: 201]. When the dark-grown prothalli were irradiated with continuous white light, marginal cells of the elongation zone divided asymmetrically, and the resulting smaller cells developed into rhizoids. The asymmetric division was also induced by brief irradiation of red light. The effect of red light was cancelled by subsequent irradiation of far-red light, indicating that the asymmetric division was regulated by phytochrome. Since the response to red light was not observed at 10(1) J m(-2) and saturated at 10(2) J m(-2) and the response is photoreversible by far-red light, the photoresponse was classified as a low-fluence response of phytochrome. Although the asymmetric division was induced by brief irradiation of red light, continuous irradiation of white, blue or red light was necessary to induce rhizoid growth. These results indicate that asymmetric division and subsequent cell growth are independently regulated by light in CERATOPTERIS: prothalli.

RSF1, an Arabidopsis locus implicated in phytochrome A signaling.

In Arabidopsis, phytochrome A (phyA) is the major photoreceptor both for high irradiance responses to far-red light and broad spectrum very low fluence responses, but little is known of its signaling pathway(s). rsf1 was isolated as a recessive mutant with reduced sensitivity to far-red inhibition of hypocotyl elongation. At the seedling stage rsf1 mutants are affected, to various degrees, in all described phyA-mediated responses. However, in adult rsf1 plants, the photoperiodic flowering response is normal. The rsf1 mutant has wild-type levels of phyA suggesting that RSF1 is required for phyA signaling rather than phyA stability or biosynthesis. RSF1 thus appears to be a major phyA signaling component in seedlings, but not in adult, Arabidopsis plants.

Sustained but Not Transient Phytochrome A Signaling Targets a Region of an Lhcb1*2 Promoter Not Necessary for Phytochrome B Action

Current evidence is inconclusive regarding the point of signaling convergence downstream from different members of the phytochrome family. In transgenic Arabidopsis, the activity of a reporter enzyme under the control of the -453 to +67 fragment of an Lhcb1*2 promoter shows very low fluence responses (VLFRs) and high-irradiance responses (HIRs) mediated by phytochrome A and low-fluence responses (LFRs) mediated by phytochrome B. A 5' deletion of the promoter to -134 abolished the HIR without affecting VLFR or LFR. In transgenic tobacco, VLFR and LFR were observed for the -176 to -31 or -134 to -31 fragments of Lhcb1*2 fused to 35S cauliflower mosaic virus minimal promoters, but only the largest fragment showed HIR. We propose that sustained activation of phytochrome A with far-red light initiates a signaling cascade that deviates from phytochrome B signaling and transient phytochrome A signaling and that this divergence extends as far as the Lhcb1*2 promoter.

Antagonistic action of low-fluence and high-irradiance modes of response of phytochrome on germination and beta-mannanase activity in Datura ferox seeds.

Seed germination is often induced by a pulse of red light perceived by phytochrome and cancelled by a subsequent pulse of far-red light. When the pulse of red light is followed by several hours of darkness, a pulse of far-red light is no longer effective and prolonged far-red is necessary to block germination. The aim was to investigate whether the red light pulse and prolonged far-red light act on the same or different processes during germination of Datura ferox seeds. Forty-five hours after the inductive red light pulse, germination could not be blocked by one pulse or six hourly pulses of far-red light, but was significantly reduced by 6 h of continuous far-red light. The pulse of red light increased embryo growth potential and the activities of beta-mannanase and beta-mannosidase extracted from the micropylar region of the endosperm. Continuous far-red light had no effect on embryo growth potential or beta-mannosidase activity, but severely reduced the activity of beta-mannanase. The effect of far-red light had the features of a high-irradiance response of phytochrome. Both germination and beta-mannanase activity were restored by a pulse of red light given after the end of the continuous far-red treatment. It is concluded that the low-fluence response and the high-irradiance response modes of phytochrome have antagonistic effects on seed germination and that the control of beta-mannanase activity is one process where this antagonism is established.

Fhy3-1 Retains Inductive Responses of Phytochrome A

The fhy3 mutation of Arabidopsis impairs phytochrome A (phyA)-mediated inhibition of hypocotyl growth without affecting the levels of phyA measured spectrophotometrically or immunochemically. We investigated whether the fhy3-1 mutation has similar effects on very low fluence responses (VLFR) and high irradiance responses (HIR) of phyA. When exposed to hourly pulses of far-red light, etiolated seedlings of the wild type or of the fhy3-1 mutant showed similar inhibition of hypocotyl growth, unfolding of the cotyledons, anthocyanin synthesis, and greening upon transfer to white light. In the wild type, continuous far-red light was significantly more effective than hourly far-red pulses (at equal total fluence). In the fhy3-1 mutant, hourly pulses were as effective as continuous far-red light, i.e. the failure of reciprocity typical of HIR was not observed. Germination was similarly promoted by continuous or pulsed far-red in wild-type and fhy3-1 seeds. Thus, for hypocotyl growth, cotyledon unfolding, greening, and seed germination, the fhy3-1 mutant retains VLFR but is severely impaired in HIR. These data are consistent with the idea that VLFR and HIR involve divergent signaling pathways of phyA.

Light-induced nuclear import of phytochrome-A:GFP fusion proteins is differentially regulated in transgenic tobacco and Arabidopsis.

Phytochromes (phy) are a family of photoreceptors that control various aspects of light-dependent plant development. Phytochrome A (phyA) is responsible for the very low fluence response (VLFR) under inductive light conditions and for the high irradiance response (HIR) under continuous far-red light. We have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco. The import is preceded by very fast, light-induced formation of sequestered areas of phyA:GFP in the cytosol. Here we report that expression of the Arabidopsis phyA:GFP fusion protein in phyA-deficient Arabidopsis plants complements the mutant phenotype. In these transgenic Arabidopsis lines, both light-dependent cytosolic formation of sequestered areas of the phyA:GFP as well as VLFR or HIR-mediated nuclear import of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein was induced only by continuous far-red light (HIR) but not by pulses of far-red light (VLFR) in transgenic tobacco. These results demonstrate that photoregulation of intracellular partitioning of the Arabidopsis phyA:GFP differs significantly in different genetic backgrounds.

Very early detection of canopy presence by seeds through perception of subtle modifications in red:far red signals

Abstract1. An early inhibition of germination in seeds of Silene gallica and Brassica campestris which were continuously exposed to the light environment under an establishing wheat canopy, was observed in two different experiments. Inhibition occurred c.15 days after crop emergence, when the canopy leaf area index (LAI) was below one and the red (R):far‐red (FR) ratio recorded under the canopy was well above 0·8.2. This inhibitory effect was either overcome by filtering FR light through a solution of CuSO4 or could be artificially imposed by simulating the canopy with filters yeilding a R:FR ratio of 0·95 and 0·8. These results show that light subtly enriched with FR was the environmental factor regulating germination below the developing canopy.3. Exposure to canopy‐filtered light pulses of 1 h (presumably sufficient to saturate a low fluence response, LFR) did not inhibit seed germination. Moreover, such treatment promotes germination up to an extent similar to that previously observed in the laboratory after a saturating pulse of R light. Instead, prolonged exposures were required to inhibit germination. These results, together with the relatively high R:FR ratios measured below the canopy in early stages of its establishment, suggest that a high irradiance response (HIR) would be involved in such a regulation.4. This capacity to detect small environmental light‐quality modifications when exposed to high irradiances, would allow the seeds from these species to detect the presence of a canopy in the very early stages of its establishment and to stay in ‘safe’ pre‐germination phases when the probability of successful seedling establishment is low.

Nuclear and cytosolic events of light-induced, phytochrome-regulated signaling in higher plants.

Plants as sessile organisms cannot walk away but have to adapt to changes in their natural environments. Light is obviously the most important external factor, being the energy source. To regulate their fitness, the photosynthetic capacity of plants must be adapted to changes in the ambient light environment. Therefore, plants have evolved a large set of photoreceptors to monitor light quality and quantity, ranging from the UV to the infrared part of the spectrum. At least three different photoreceptor classes have been identified and analyzed: (i) UV‐B receptors, primarily characterized by action spectroscopy (Wellmann, 1983); (ii) the blue UV‐A photoreceptors, cry1 and cry2 and phototropine (Ahmad and Cashmore, 1993; Christie et al ., 1998; Lin et al ., 1998); and (iii) the red/far‐red reversible phytochromes. These photoreceptors control different aspects of plant growth and development, and in this review we focus on the physiological and molecular events regulated by the most characterized plant photoreceptors, the phytochromes. In higher plants, phytochromes are encoded by a small multigene family; five genes ( PHYA–PHYE , Sharrock and Quail, 1989; Clack et al ., 1994) have been identified in Arabidopsis . Phytochromes exist as dimers composed of two 125 kDa polypeptides, each carrying a covalently linked tetrapyrrol chromophore in the N‐terminal domain and dimerization domains in the C‐terminal domain. The photosensory function of the molecule is based on its capacity for reversible interconversion between the red light‐absorbing Pr form and the far‐red light absorbing Pfr form following sequential absorption of red and far‐red light. Photosignal perception by the receptor is followed by conformational changes, and activates, through an as yet poorly understood mechanism, signaling pathways leading to changes in the expression of genes that underlie developmental responses to light (Quail et al ., 1995). In order to finely tune their …

Elementary processes of photoperception by phytochrome A for high-irradiance response of hypocotyl elongation in Arabidopsis.

Elementary processes of photoperception by phytochrome A (PhyA) for the high-irradiance response (HIR) of hypocotyl elongation in Arabidopsis were examined using a newly designed irradiator with LED. The effect of continuous irradiation with far-red (FR) light could be replaced by intermittent irradiation with FR light pulses if given at intervals of 3 min or less for 24 h. In this response, the Bunsen-Roscoe law of reciprocity held in each FR light pulse. Therefore, we determined the action spectrum for the response by intermittent irradiation using phyB and phyAphyB double mutants. The resultant action spectrum correlated well with the absorption spectrum of PhyA in far-red-absorbing phytochrome (Pfr). Intermittent irradiation with 550 to 667 nm of light alone had no significant effect on the response. In contrast, intermittent irradiation with red light immediately after each FR light pulse completely reversed the effect of FR light in each cycle. The results indicate that neither red-absorbing phytochrome synthesized in darkness nor photoconverted Pfr are physiologically active, and that a short-lived signal is induced during photoconversion from Pfr to red-absorbing phytochrome. The mode of photoperception by PhyA for HIR is essentially different from that by PhyA for very-low-fluence responses and phytochrome B for low-fluence responses.

Contrasting seed germination responses to red and far-red light in Leptospermum scoparium and Melicytus ramiflorus

Effects of red light (R) and far-red light (FR), and selected photon flux densities (PFD) of photosynthetically active radiation (PAR) on seed germination in the photoblastic, primary colonising species Leptospermum scoparium J. R. et G. Forst. and the late secondary successional Melicytus ramiflorus J. R. et G. Forst. were studied. A continuous R dose response curve forL. scoparium germination was developed, unifying data from experiments using long-term exposure to PAR with those following short-term exposure to R. The threshold R dose needed to effect germination was ~0.1 mmol m –2 , and the response was saturated at 1000 mmol m –2 . Stimulation of germination by R was reversed by a subsequent exposure to FR. These features are consistent with a low-fluence response mediated by phytochrome B. FR reversal of germination was achieved at a dose two orders of magnitude lower than that of R required to induce initial germination. However, when both R and FR were provided simultaneously, the FR dose needed to even partially inhibit germination (34% compared to > 95% in controls) was two orders of magnitude higher than the R dose (R:FR ratio = 0.007). Germination in L. scoparium was also stimulated in up to 12% of seed upon diurnal exposure to FR, or by green light (~2 mol m –2 ), indicating a very-low-fluence response mediated by phytochrome A also operating in this species. In contrast, seed germination in M. ramiflorus was relatively unresponsive to R, and secondary dormancy was induced by high PFD (515 mol m –2 s –1 ).

Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B

The phytochrome (phy) family of plant photoreceptors controls various aspects of photomorphogenesis. Overexpression of rice phyA-green fluorescent protein (GFP) and tobacco phyB-GFP fusion proteins in tobacco results in functional photoreceptors. phyA-GFP and phyB-GFP are localized in the cytosol of dark-adapted plants. In our experiments, red light treatment led to nuclear translocation of phyA-GFP and phyB-GFP, albeit with different kinetics. Red light-induced nuclear import of phyB-GFP, but not that of phyA-GFP, was inhibited by far-red light. Far-red light alone only induced nuclear translocation of phyA-GFP. These observations indicate that nuclear import of phyA-GFP is controlled by a very low fluence response, whereas translocation of phyB-GFP is regulated by a low fluence response of phytochrome. Thus, light-regulated nucleocytoplasmic partitioning of phyA and phyB is a major step in phytochrome signaling.

Regulation of phytochrome B signaling by phytochrome A and FHY1 in Arabidopsis thaliana.

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very-low fluence responses (VLFR) induced by pulsed far-red light and the high-irradiance responses (HIR) observed under continuous far-red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low-fluence responses (LFR) induced by red-light pulses and reversed by subsequent far-red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB-mediated responses. However, a pre-treatment with continuous far-red light enhanced the LFR induced by a subsequent red-light pulse. This enhancement was absent in phyA, phyB, or phyA phyB and partial in fhy1. The levels of phyB were not affected by the phyA or fhy1 mutations or by far-red light pre-treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far-red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.

Phytochrome elicits the cryptic red-light signal which results in amplification of anthocyanin biosynthesis in sorghum

Anthocyanin synthesis in Sorghum bicolor Moench induced by a low-fluence response of phytochrome (phy) is multiplicatively amplified by a cryptic red-light signal (CRS) produced by red light (R). The photoreceptor for CRS and its features in CRS production were studied. (i) An action spectrum determined with a 200-s light pulse of wavelengths from 347 to 693 nm had peaks at 657 and 378 nm. (ii) The CRS-producing effect of R, even as short a pulse as 20 s, was neither suppressed by an immediately subsequent far-red light (FR) pulse nor increased by placing a dark interval of 180 s between R and FR; simultaneous FR, however, suppressed the R action in accordance with the resulting ratios of the FR-absorbing form (Pfr) to total phy. (iii) The effect of R increased with increasing fluence rate to plateau at the same fluence rate regardless of the pulse length, but the level of this plateau depended on the pulse length. (iv) The effect of R increased with increasing pulse length when compared at the same fluence, whether saturating or unsaturating; thus, no reciprocity law holds. These results indicate that the photoreceptor for CRS production is a phy, Pfr being active, which presumably shows very fast dark reversion to the R-absorbing form without absorbing FR. The possible CRS-production mechanism of the phy and its significance in the so-called R high-irradiance response of phy are discussed.