Tomato seed germination: regulation of different response modes by phytochrome B2 and phytochrome A

Lycopersicon esculentum seeds germinate after rehydration in complete darkness. This response was inhibited by a far-red light (FR) pulse, and the inhibition was reversed by a red light (R) pulse. Comparison of germination in phytochrome-deficient mutants (phyA, phyB1, phyB2, phyAB1, phyB1B2 and phyAB1B2) showed that phytochrome B2 (PhyB2) mediates both responses. The germination was inhibited by strong continuous R (38 micromol m(-2) s(-1)), whereas weak R (28 nmol m(-2) s(-1)) stimulated seed germination. Hourly applied R pulses of the same photon fluence partially replaced the effect of strong continuous R. This response was called 'antagonistic' because it counteracts the low fluence response (LFR) induced by a single R pulse. This antagonistic response might be an adaptation to a situation where the seeds sit on the soil surface in full sunlight (adverse for germination), while weak R might reflect that situation under a layer of soil. Unexpectedly, the effects of continuous R or repeated R pulses were mediated by phytochrome A (PhyA). We therefore suggest that low levels of PhyA in its FR-absorbing form (Pfr) cause inhibition of seed germination produced either by extended R irradiation (by degradation of PhyA-Pfr) or by extended FR irradiation [keeping a low Pfr/R-absorbing form (Pr) ratio].     

Biphasic fluence-response curves for light induced germination of Arabidopsis thaliana seeds

Abstract With appropriate pretreatment of the seeds fluence-response curves for the induction of germination of Arabidopsis thaliana show two phases. A proportion of the population responds to very low fluence (VLFR), 10⁴–10^?2μmolm^?2 establishing 10^?4–10^?2% of the total phytochrome in the far-red absorbing form (Pfr) and a proportion of the population respond to low fluence (LFR), 1–1000 μmolm^?2, establishing 1–75% Pfr. The VLFR is nol normally seen because the pre-existing Pfr level satisfies the Pfr requirement or use of green safelight establishes more Pfr than necessary to saturate the VLFR. Endogenous Pfr was depicted by a 24 h 35°C treatment, presumably as a result of dark destruction and/or dark reversion to the red absorbing form of phytochrome (Pr), making it possible to visualize the VLFR. A short pulse of 35°C treatment in combination with an appropriate temperature regime is also able to sensitize a proportion of the seed population. The proportion of the population showing the VLFR is determined by the duration of the cold imbibition pretreatment as well as the duration of the 35°C treatment. Complex fluence-response curves were observed in which a proportion of the seeds being promoted in the VLFR range, were inhibited at higher fluences before being further promoted in the LFR range. This was particularly clear for seed batches being sensitized by a short 35°C treatment. The VLFR may be of significance in the natural environment, enabling seeds buried in the upper layer of the soil to germinate, where the fluence rate falls off sharply and the LFR is not satisfied. A model is presented to explain the two phases in the fluence-response curves.     

The very-low-fluence and high-irradiance responses of the phytochromes have antagonistic effects on germination, mannan-degrading activities, and DfGA3ox transcript levels in Datura ferox seeds

Seed germination can be promoted by the modes of action of two of the phytochromes: the low-fluence response (LFR), which is the classical red (R)-far-red (FR) reversible response and the very-low-fluence response (VLFR) that can be saturated by extremely low levels of Pfr, which can be elicited by a saturating FR pulse. The Datura ferox seed population used in this work had acquired the capacity to germinate through a VLFR after pretreatment in a water-saturated atmosphere (WSA) at constant 25 degrees C. After 12 d in WSA germination after a FR pulse was 82%, while it was less than 10% in darkness. It was found that the VLFR of germination is associated with increments in the embryo growth potential (EGP) and in the activity of two enzymes related to the weakening of the micropylar region of the endosperm (ME); endo-beta-mannanase and beta-mannosidase. The FR pulse also significantly stimulated the expression of DfGA3ox, a GA 3beta-hydroxylase, suggesting that the promotion of germination by the VLFR is associated with an increase in the synthesis of active gibberellins. The promotive action of the VLFR on germination is reduced when the FR pulse is immediately followed by a continuous FR treatment for 24 h (FRc). The effect of FRc cannot be reproduced by hourly FR pulses during the same period, showing that the antagonistic effect of FRc is a high-irradiance response (HIR). The action of the HIR in germination is associated with a decrease of both the mannan-degrading enzyme activity and the expression of DfMan in the ME, whereas no changes in the EGP were observed. The HIR also inhibits the accumulation of DfGA3ox in embryos, indicating that its action on germination is mediated, at least in part, through the modulation of active GA contents in seeds. This is the first report of a gene that participates in the VLFR-HIR antagonism in seeds.     

Light environment in pre- and post-dehiscent fruits affects seed germination in Calotropis procera

Fruits in Calotropis procera can be distinguished into five discrete but contiguous stages on the basis of diameter and seed color. Seeds from dehisced fruits at stage V germinated >80% on moist substratum in darkness. This was rather unexpected because the seeds developed and matured in an FR-enriched microenvironment (R:FR ratio ～0.3) of the chlorophyll-containing maternal tissue and displayed low-fluence response (LFR) mode of phytochrome action. In contrast to >80% dark-germinating seeds from dehisced fruits at stage V, about 50% seeds from undehisced fruits at that stage were dark germinating, whereas another 30% seeds required light for germination. The light-requiring fraction of the seed population did not only respond to a very low-fluence R and to a short FR pulse, but also lacked R–FR reversibility thereby indicating to a very low-fluence response (VLFR) mode of phytochrome action. The present study reporting VLFR to non-dormant seed state transition in C. procera suggested that the state of phytochrome and the subsequent seed germination response in dry-seeded species, besides being determined by the light environment immediately before maturation drying, might also be regulated by a post-dehiscence light signal.     

Phytochrome regulation of seed germination

Seed germination of many plant species is influenced by light. Of the various photoreceptor systems, phytochrome plays an especially important role in seed germination. The existence of at least five phytochrome genes has led to the proposal that different members of the family have different roles in the photoregulation of seed germination. Physiological analysis of seed germination ofArabidopsis thaliana (L.) Heynh. with phytochrome-deficient mutants showed for the first time that phytochrome A and phytochrome B modulate the timing of seed germination in distinct actions. Phytochrome A photo-irreversibly triggers the photoinduction of seed germination after irradiation with extremely low fluence light in a wide range of wavelengths, from UV-A, to visible, to far-red. In contrast, phytochrome B mediates the well-characterized photoreversible reaction, responding to red and far-red light of fluences four orders of magnitude higher than those to which PhyA responds. Wild plants, such asA. thaliana, survive under ground as dormant seeds for long periods, and the timing of seed germination is crucial for optimizing growth and reproduction. It therefore seems reasonable for plants to possess at least two different physiological systems for sensing the light environment over a wide spectral range with exquisite sensitivity of different phytochromes. This redundancy seems to enhance plant survival in a fluctuating environment.     

Photosensitivity of Rumex obtusifolius seeds for stimulation of germination: Influence of light and temperature

A population of Rumex obtusifolius L. seeds imbibed for 24 h at 25°C exhibits a sigmoid logarithmic fluence-response relationship for stimulation of germination by red light (R), 11.0 μmol m⁻² being necessary for 50% of the response. After 24 h imbibition at 35°C the fluence-response relationship for stimulation of germination by R is biphasic. For 50% response the very sensitive phase (very low fluence-response) requires 4.7 − 10⁻²μmol m⁻² whereas the less sensitive phase (low fluence-response) requires 4.0 μmol m². A few seconds of far-red light (FR) satisfies the germination requirement of the sensitive seeds after 24 h at 35°C. However, a longer period of FR (2 h) results in low germination. The fluence-response relationship for induction of these seeds by R is sigmoid, 4.8 μmol m⁻² being necessary for 50% response, demonstrating that 2 h FR desensitizes the sensitive proportion of the seed population induced by 24 h at 35°C. A proportion of the seed population can be further sensitized by 60 min at 35°C following this desensitization.     

Avena phytochrome a overexpressed in transgenic tobacco seedlings differentially affects red/far-red reversible and very-low-fluence responses (cotyledon unfolding) during de-etiolation

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.Abbreviations FR far-red light - LFR low-fluence response - Pfr/P proportion of phytochrome (P) in its FR-absorbing form (Pfr) - R red light - VLFR very low-fluence response Financial support was provided by the University of Buenos Aires and Fundación Antorchas (Argentina) to J.J.C., CONICET (Argentina) to R.A.S. and the U.S. Department of Energy (DE-FG02-88ER13968) to R.D.V.     

Changes in the light sensitivity of buried Polygonum aviculare seeds in relation to cold-induced dormancy loss: development of a predictive model

The effect of cold (stratification) temperature on changes in the sensitivity of Polygonum aviculare seeds to light was investigated. Seeds buried in pots were stored under stratification temperatures (1.6, 7 and 12 degrees C) for 137 d. Seeds exhumed at regular intervals during storage were exposed to different light treatments. Germination responses obtained for seeds exposed to different light treatments and stratification temperatures were used to develop a model to predict the sensitivity of buried seeds to light. Seed sensitivity to light increased as dormancy loss progressed, showing the successive acquisition of low-fluence responses (LFR), very low-fluence responses (VLFR), and the loss of the light requirement for germination for a fraction of the seed population. These changes were inversely correlated to stratification temperature, allowing the use of a thermal time index to relate observed changes in seed light sensitivity to stratification temperature. The rate of increase in sensitivity of P. aviculare seeds to light during stratification is inversely correlated to soil temperature, and these changes in light sensitivity could be predicted in relation to temperature using thermal-time models.     

Brassinosteroid Mutants Uncover Fine Tuning of Phytochrome Signaling

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.     

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 ( VLF1 ) and another on chromosome 5 ( VLF2 ), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.     

Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.     

Fluence-response curves and action spectra for the very low fluence and the low fluence response for the induction of kalanchoë seed germination

Germination of Kalanchoë blossfeldiana Poelln. seeds is absolutely light-requiring. Germination of one seed is the result of one out of three reactions, viz. the very low fluence response (VLFR), the low fluence response (LFR) and the high fluence response/high irradiance response. In order to demonstrate the involvement of phytochrome for both photoresponses, i.e. VLFR and LFR, action spectra for induction were determined. Fluence-response data are analyzed by means of probit analysis in order to calculate the seed population parameters, with special attention to μ, or the fluence for half-maximal induction, and B, the slope in the probit diagram. Laser light was used between 620 and 800 nanometers to analyze the VLFR. Phytochrome is responsible for both photoresponses: the VLFR action spectrum demonstrates an exponential decrease in apparent photoconversion cross-section (P_r → P_fr) up to about 800 nanometers. Assuming that P_r:P_fr-X and P_fr:P_fr-X are the effectors for the VLFR and the LFR, respectively, we estimate an average induction threshold of about 0.003% P_r:P_fr for the VLFR and about 1% P_fr:P_fr for the LFR among individuals of the seed population.     

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.     

Factors affecting the induction and release of secondary dormancy in Kalanchoë seeds

Abstract. Several short daily R irradiations are required from the first day of incubation on water to induce germination of Kalanchoë seeds. When the same light treatment is given after a prolonged dark incubation period at 20°C, secondary dormancy prevents germination. Factors controlling the induction and breaking of secondary dormancy have been investigated. The induction of secondary dormancy is very temperature dependent. Locally puncturing the seed coat strongly delays it. Secondary dormancy is not induced in the presence of GA₃ during the first 10 d of dark incubation, although this growth substance cannot induce dark germination. Prolonged or cyclic daily R irradiations can relieve secondary dormancy of seeds kept on water, even after a dark period of 20 d. A 24 h treatment at 4°C restores responsiveness to short R exposures of slightly secondarily dormant seeds. The synergism between GA₃ and Pfr in non-dormant Kalanchoë seeds, leading to high effectiveness of even one short FR irradiation, still occurs in seeds made secondarily dormant before transfer to GA₃, but more R or FR irradiations, in combination with GA₃, are required for the release of secondary dormancy. A combination of red light and 6-benzyl-aminopurine is ineffective in removing dormancy.     

Analysis of the Effect of Light and Temperature on the Fluence Response Curves for Germination of Rumex obtusifolius

Both red light (10 minutes) and 35°C treatment (60 minutes) stimulate the germination of seeds of Rumex obtusifolius otherwise maintained in darkness at 25°C. Fluence response curves were determined for the effect of red light to stimulate germination of seeds with and without 35°C treatment. The endogenous far-red absorbing form (Pfr) level in the seeds was determined using short saturating fluences of wavelengths of light which maintain different proportions of phytochrome as Pfr at equilibrium. In the seed batches investigated, the endogenous Pfr level was found to be 4% or less of the total phytochrome. High dark germination after 35°C treatment does not result from an increase in sensitivity of the whole population to Pfr. Calculated fluence response curves for germination which best fit the experimental data suggest that seeds germinate in darkness after 35°C treatment because of a nonphytochrome-related process (overriding factor).     

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma

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.     

Sensitivity of Polygonum aviculare seeds to light as affected by soil moisture conditions

BACKGROUND AND AIMS: It has been hypothesized that soil moisture conditions could affect the dormancy status of buried weed seeds, and, consequently, their sensitivity to light stimuli. In this study, an investigation is made of the effect of different soil moisture conditions during cold-induced dormancy loss on changes in the sensitivity of Polygonum aviculare seeds to light. METHODS: Seeds buried in pots were stored under different constant and fluctuating soil moisture environments at dormancy-releasing temperatures. Seeds were exhumed at regular intervals during storage and were exposed to different light treatments. Changes in the germination response of seeds to light treatments during storage under the different moisture environments were compared in order to determine the effect of soil moisture on the sensitivity to light of P. aviculare seeds. KEY RESULTS: Seed acquisition of low-fluence responses during dormancy release was not affected by either soil moisture fluctuations or different constant soil moisture contents. On the contrary, different soil moisture environments affected seed acquisition of very low fluence responses and the capacity of seeds to germinate in the dark. CONCLUSIONS: The results indicate that under field conditions, the sensitivity to light of buried weed seeds could be affected by the soil moisture environment experienced during the dormancy release season, and this could affect their emergence pattern.     

Light exaggerates apical hook curvature through phytochrome actions in tomato seedlings

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.     

Intramolecular uncoupling of chromophore photoconversion from structural signaling determinants drive mutant phytochrome B photoreceptor to far-red light perception

The phytochrome (phy) photoreceptor family regulates almost all aspects of plant development in a broad range of light environments including seed germination, onset of the photomorphogenic program in seedling stage, the shade avoidance syndrome in competing plant communities, flowering induction and senescence of adult plants. During evolution two clearly distinct classes of phy-s emerged covering these very different physiological tasks.¹ PhyA is rapidly degraded in its activated state. PhyA functions in controlling seed germination at very low light intensities (very low fluence response, VLFR) and seedling establishment under photosynthetic shade conditions (high irradiance response, HIR) where the far-red portion of the transmitted light to understorey habitats is substantially enhanced. Arabidopsis phyB together with phyC, D and E belongs to the relatively stable sensor class in comparison to the light labile phyA. PhyB functions at all stages of development including seed germination and seedling establishment, mediates classical red/far-red reversible low fluence responses (LFR) as well as red light high irradiance responses, and it is considered to be the dominating phytochrome sensor of its class.