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.     

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.     

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.     

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

Phytochrome intermediates and action spectra for light perception by dry seeds

It has previously been demonstrated that far-red irradiation of dry Lactuca sativa L. seeds results in inhibition of subsequent germination. Although red has no effect on dry seeds, a red irradiation following a farred irradiation reverses the effect of far-red. This phenomenon is most noticeable in seeds with artificially raised levels of phytochrome in the far-red absorbing form. Qualitatively similar results have been found for the seeds of Plantago major L., Sinapis arvensis L., and Bromus sterilis L. Action spectra studies on Plantago seeds show that the action peaks for promotion and inhibition of germination of hydrated seeds are at 660 and 730 nanometers, respectively. The action spectrum for inhibition of subsequent germination following irradiation of dry seeds is qualitatively and quantitatively similar to that for hydrated seeds, with an action peak at 730 nanometers, indicating absorption by phytochrome in the far-red absorbing form. However, the action spectrum for the reversal of this far-red effect on dry seeds has a broad peak at 680 nanometers and subsidiary peaks at 650 and 600 nanometers. It is proposed that this effect is due to light absorption by the phytochrome intermediate complex meta-Fa, and that the action spectrum reflects the in vivo absorption properties of this intermediate.     

Phytochrome-mediated growth inhibition of seminal roots in rice seedlings

In rice ( Oryza sativa ) seedlings, continuous white-light irradiation inhibited the growth of seminal roots but promoted the growth of crown roots. In this study, we examined the mechanisms of photoinhibition of seminal root growth. Photoinhibition occurred in the absence of nitrogen but increased with increasing nitrogen concentrations. In the presence of nitrogen, photoinhibition was correlated with coiling of the root tips. The seminal roots were most photosensitive 48–72 h after germination during the 7-day period after germination. White-light irradiation for at least 6 h was required for photoinhibition, and the Bunsen–Roscoe law of reciprocity was not observed. Experiments with phytochrome mutants showed that far-red light was perceived exclusively by phyA, red light was perceived by both phyA and phyB, and phyC had little or no role in growth inhibition or coiling of the seminal roots. These results also suggest that other blue-light photoreceptors are involved in growth inhibition of the seminal roots. Fluence-response curve analyses showed that phyA and phyB control very low-fluence response and low-fluence response, respectively, in the seminal roots. This was essentially the same as the growth inhibition previously observed at the late stage of coleoptile development (80 h after germination). The photoperceptive site for the root growth inhibition appeared to be the roots themselves. All three phytochrome species of rice were detected immunochemically in roots.     

Phytochrome Action during Prechilling Induced Germination of Betula papyrifera Marsh

Seeds of paper birch (Betula papyrifera Marsh.) were induced to germinate by prechilling at 3 C or by red light. The light requirement was mediated by phytochrome and the action of phytochrome during prechilling was investigated. Red irradiation (R) prior to prechilling markedly enhanced the effectiveness of the prechilling treatment in inducing subsequent germination at 18 C. Reversal of this enhancement by far-red irradiation (FR) was more effective when FR was supplied after a 1-week prechill treatment than after a 2-week treatment. The R enhancement effect exhibited a sharp drop as prechilling temperature was increased from 5 to 7 C. This decline is consistent with a membrane phase transition at about 7 C where Pfr action is diminished by a loss in sensitivity of its receptor sites. Although phytochrome action was observed during prechilling treatments, the seeds failed to germinate at prechilling temperatures. Therefore, it was concluded that while potentiation of germination by Pfr occurred during prechilling, some other reaction(s) leading to radicle protrusion requires higher temperatures. In one seed source loss of germination potential was observed with protracted storage at 3 C. This was prevented by R supplied during the prechilling treatment. Taken collectively the data suggest that action of phytochrome during prechilling is accentuated in these seeds by two factors: (a) an increase in the sensitivity (or number) of Pfr receptor sites; and (b) preservation of Pfr by deferment of thermal reversion.     

Photocontrol of the germination of onoclea spores: I. Action spectrum

Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm⁻²; sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.     

Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings

The action spectrum for the light-activated destruction of phytochrome in etiolated Avena seedlings has been determined. There are 2 broad maxima, one between 380 and 440 mμ, the other between 600 and 700 mμ. peaking at about 660 mμ. On an incident energy basis, the red region of the spectrum is more efficient than the blue by about one order of magnitude in activating phytochrome disappearance. Both the red absorbing as well as the far-red absorbing forms of phytochrome are destroyed after exposure of Avena seedling to either red or blue light.

From the action spectrum and photoreversibility of pigment loss, we conclude that phytochrome acts as a photoreceptor for the photoactivation of its metabolically-based destruction. We suggest that another pigment might also be associated with the disappearance of phytochrome in oat seedlings exposed to blue light.

     

Studies on the mechanism of light-dependent germination of akinetes of blue-green algae

This paper deals with the role of light in the germination of akinetes of Anabaena azollae. The two maxima action spectra are situated at 385 and 615 nm and the stimulation of the germination process by photosynthate was confirmed. The photoreceptor absorbing at 385 nm was identified as a flavin and that at 615 nm as a phytochrome. A model is suggested for the mode of action of light in the germination of akinetes of blue-green algae.C. Tsui     

The role of phytochrome in an interaction with ethylene and carbon dioxide in overcoming lettuce seed thermodormancy

Ethylene and CO₂ were used to control induction of germination in thermodormant lettuce seed (Lactuca sativa L.). These experiments ultimately showed that germination depends on the presence of an active form of the phytochrome. The phytochrome system is functional and stable at 35 C, a temperature which completely inhibits germination. Phytochrome responses to red or far red light and darkness showed that this inhibition of germination under light must be due to some other block(s) rather than to a direct inactivation of the phytochrome system itself. A postred radiation increase in lettuce seed germination that is not reversed by far red light was observed. The CO₂ requirement for C₂H₄ action is not due to a change in the medium's pH; addition of C₂H₄ plus CO₂ at the start of imbibition did not result in as much germination as when they were added several hours after imbibition. This reduction in germination, when the gases are added at the start of imbibiton, is due to CO₂.     

Opposing Actions of Light in Seed Germination of Poa pratensis and Amaranthus arenicola

Action spectra were measured for suppression of germination of Poa pratensis L. and Amaranthus arenicola I. M. Johnston seed under prolonged or continuous irradiation. The action maxima for both types of seeds are near 720 nm. The maxima are unchanged in position or magnitude in the presence of radiation in the region of 600 to 670 nm adequate to maintain phytochrome predominantly in the far-red-absorbing form. A reversible potentiation of germination to change in form of phytochrome was observed for both seeds. The bearing of these findings on a high-energy regulatory light response is discussed.     

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.     

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.     

Photomorphogenic mutants of Arabidopsis thaliana reveal activities of multiple photosensory systems during light-stimulated apical-hook opening

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.We thank Jeff Young and Brian Parks for their many helpful suggestions during the progress of this research. This work was supported by National Science Foundation Grant No. DCB-9106697.     

Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes

An action spectrum for anthocyanin formation in dark-grown broom sorghum (Sorghum bicolor Moench, cv Acme Broomcorn and cv Sekishokuzairai Fukuyama Broomcorn) seedlings was determined over the wavelength range from 260 to 735 nanometers. The action peaks were at 290, 650, 385, and 480 nanometers in descending order of height. The action of the 290-nanometer peak was not affected by subsequently given far red light, whereas those of the other three action peaks were nullified completely. The nullification of the 385-nanometer peak action by far red light was reversible. When an irradiation at these action peaks was followed by a phytochrome-saturating fluence of red light irradiation, the action of the 290-nanometer peak remained, whereas that of the 385-nanometer peak as well as those of the 650- and 480-nanometer peaks was masked by the action of the second irradiation. These findings suggested that the 290- and 385-nanometer action peaks involved different photoreceptors, the latter being phytochrome. The blue light-absorbing photoreceptor as reported to be a prerequisite for phytochrome action in milo sorghum was not found to exist in the broom sorghums.

The action spectrum deprived of the involvement of phytochrome was determined in the ultraviolet region by irradiating with far red light following monochromatic ultraviolet light. The spectrum had a single intense peak at 290 nanometers and no action at all at wavelengths longer than 350 nanometers.

     

Light-induced Ethylene Production in Sorghum

Ethylene production was induced in sections of dark-grown Sorghum vulgare L. seedlings by treatment with light in the blue and far red regions of the light spectrum. The action spectrum closely resembled the previously reported spectra for high irradiance response; thus, light-induced ethylene production is probably a high irradiance response with phytochrome as the initial photoreceptor.     

Germination in spores of Dryopteris filix-mas: Regulation of rhizoid elongation as a second phytochrome-mediated response

Spore germination in Dryopteris filix-mas occurs via a cascade of cellular responses, and chlorophyll formation, mitosis or rhizoid elongation are commonly used as parameters to determine spore germination. Detailed investigations of these parameters led to the hypothesis that they are regulated by different, independent phytochrome-mediated responses. This concept could be confirmed, as is described in this paper which demonstrates that perception of light via phytochrome occurs within two different phases separated in time. Presence of the far-red absorbing phytochrome form, P_fr, for 36 h, induces chlorophyll formation and the first unequal cell division, by which a rhizoid initial and a protonemal initial are formed (first phytochrome-mediated response). However, rhizoid elongation requires a second period of P_fr, presence (second phytochrome-mediated response). There is a clear temporal distinction between the first and the second phytochrome-mediated response with respect to the coupling of P_fr to the transduction chain; P_fr is unable to induce rhizoid growth until 60 h after the start of the first red irradiation. The effectivity of P_fr for inducing the second response shows an optimum at ca 96 h after the beginning of the presence of P_fr; thereafter, it declines slowly. The fluence-response relationship and the presence of red/far-red reversibility demonstrate that rhizoid elongation is a low-fluence response mediated by phytochrome and is independent of the first phytochrome response.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.