Isolation and Initial Characterization of Arabidopsis Mutants That Are Deficient in Phytochrome A

Phytochrome, a red/far-red-light photoreceptor protein of plants, is encoded by a small gene family. Phytochrome A (PHYA), the product of the PHYA gene, is the predominant molecular species of phytochrome in etiolated tissue and has been best characterized biochemically. To define a role for PHYA, we isolated new mutants, designated fre1 (far-red elongated), in Arabidopsis thaliana that were specifically deficient in PHYA spectral activity and protein accumulation. These mutants were identified on the basis of their long hypocotyl phenotype under continuous far-red light. Although the fre1 mutants lacked the hypocotyl response to continuous far-red light, their responses to continuous white light and to end-of-day far-red-light treatments were normal. Thus, PHYA appears to play only a minor role in the regulation of hypocotyl elongation under natural conditions. In contrast, the fre1 mutation affected greening a fre1 mutant was less able than the wild type to deetiolate after growth in the dark. However, the potentiation effect of a red-light pulse on accumulation of chlorophyll was not changed significantly in the fre1 mutants. Thus, the function of PHYA might be highly specialized and restricted to certain phases of Arabidopsis development.     

Photophysiology and phytochrome content of long-hypocotyl mutant and wild-type cucumber seedlings

Photomorphogenetic responses have been studied in a cucumber (Cucumis sativus L.) mutant (lh), which has long hypocotyls in white light (WL). While etiolated seedlings of this mutant have a similar phytochrome content and control of hypocotyl elongation as wild type, deetiolation is retarded and WL-grown seedlings show reduced phytochrome control. Spectrophotometric measurements exhibit that WL-grown tissues of the lh mutant (flower petals and Norflurazon-bleached leaves) contain 35 to 50% of the phytochrome level in the wild type. We propose that this is a consequence of a lack of light-stable phytochrome, in agreement with our hypothesis proposed on the basis of physiological experiments. The lh mutant lacks an end-of-day far-red light response of hypocotyl elongation. This enables the end-of-day far-red light response, clearly shown by the wild type, to be ascribed to the phytochrome, deficient in the lh mutant. Growth experiments in continuous blue light (BL) and continuous BL + red light (RL) show that when RL is added to BL, hypocotyl growth remains inhibited in the wild type, whereas the lh mutant exhibits significant growth promotion compared to BL alone. It is proposed that the hypocotyls fail to grow long in low fluence rate BL because photosynthesis is insufficient to sustain growth.     

Photocontrol of petiole elongation in light-grown strawberry plants

Summary The mode of phytochrome control of elongation growth was studied in fully-green strawberry (Fragaria x Ananassa Duch.) plants. Petiole growth showed two distinct types of response to light. In one, the end-of-day response, petioles were lengthened by low-intensity far-red irradiation for 1 h immediately following the 8 h photoperiod. The response was little or no greater with prolonged exposure and less when the start of far-red was delayed. It was already evident in the first leaf to emerge after treatment began. With the development of successive leaves a second, photoperiodic, type of response appeared, in which petioles lengthened following only prolonged exposure to red, far-red, mixtures of the two, or tungsten lighting, all at low levels of intensity. As with the inhibition of flowering in previous experiments, irradiation with red light during the second half of the otherwise long dark period gave the greatest response.Abbreviations and Symbols FR far-red light - HIR high irradiance response - R red light - P_r phytochrome in the red light absorbing form - P_fr phytochrome in the far-red light absorbing form - SDP short-day plant - LDP long-day plant - PAR photosynthetically active radiation     

Function of phytochrome A in potato plants as revealed through the study of transgenic plants.

We have generated transgenic potato plants (Solanum tuberosum) containing the potato phytochrome protein encoded by the PHYA gene cDNA (phyA) in sense or antisense orientation under the control of the 35S cauliflower mosaic virus promoter. Plants with increased and decreased phyA levels were analyzed. When grown under white light, development and growth of sprouts and plants were barely distinguishable from wild type. Under continuous far-red light, stem extension, leaf expansion, and hook opening of sprouts were accelerated in phyA overexpressors and delayed in antisense plants. Sprouts with reduced phyA levels were less sensitive to red light with regard to stem extension and expression of the small subunit genes for ribulose bisphosphate carboxylase. Under low red light:far-red light ratios, increased phyA levels reduced the stem extension component of the shade-avoidance response, whereas decreased levels led to an increase in the response.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.     

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.     

Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Phytochrome,Gibberellins, and Hypocotyl Growth (A Study Using the Cucumber (Cucumis sativus L.) long hypocotyl Mutant)

The possible involvement of gibberellins (GAs) in the regulation of hypocotyl elongation by phytochrome was examined. Under white light the tall long hypocotyl (lh) cucumber (Cucumis sativus L.) mutant, deficient in a type B-like phytochrome, shows an increased "responsiveness" (defined as response capability) to applied GA4 (the main endogenous active GA) compared to the wild type. Supplementing far-red irradiation results in a similar increase in responsiveness in the wild type. Experiments involving application of the precursor GA9 and of an inhibitor of GA4 inactivation suggest that both the GA4 activation and inactivation steps are phytochrome independent. Endogenous GA levels of whole seedlings were analyzed by combined gas chromatography-mass spectrometry using deuterated internal standards. The levels of GA4 (and those of GA34, the inactivated GA4) were lower in the lh mutant under low-irradiance fluorescent light compared with the wild type, similar to wild type under higher irradiance light during the initial hypocotyl extension phase, and higher during the phase of sustained growth, in which extension involved an increase in the number of cells in the upper region. In all cases, growth of the lh mutant was more rapid than that of the wild type. It is proposed that GA4 and phytochrome control cell elongation primarily through separate mechanisms that interact at a step close to the terminal response.     

Overexpression of rice phytochrome A partially complements phytochrome B deficiency in Arabidopsis

The red/far-red reversible phytochromes play a central role in regulating the development of plants in relation to their light environment. Studies on the roles of different members of the phytochrome family have mainly focused on light-labile, phytochrome A and light-stable, phytochrome B. Although these two phytochromes often regulate identical responses, they appear to have discrete photosensory functions. Thus, phytochrome A predominantly mediates responses to prolonged far-red light, as well as acting in a non-red/far-red-reversible manner in controlling responses to light pulses. In contrast, phytochrome B mediates responses to prolonged red light and acts photoreversibly under light-pulse conditions. However, it has been reported that rice (Oryza sativa L.) phytochrome A operates in a classical red/far-red reversible fashion following its expression in transgenic tobacco plants. Thus, it was of interest to determine whether transgenic rice phytochrome A could substitute for loss of phytochrome B in phyB mutants of Arabidopsis thaliana (L.) Heynh. We have observed that ectopic expression of rice phytochrome A can correct the reduced sensitivity of phyB hypocotyls to red light and restore their response to end-of-day far-red treatments. The latter is widely regarded as a hallmark of phytochrome B action. However, although transgenic rice phytochrome A can correct other aspects of elongation growth in the phyB mutant it does not restore other responses to end-of-day far-red treatments nor does it restore responses to low red:far-red ratio. Furthermore, transgenic rice phytochrome A does not correct the early-flowering phenotype of phyB seedlings. Received: 12 July 1998 / Accepted: 13 August 1998     

A photoperiod-insensitive barley line contains a light-labile phytochrome B

Barley (Hordeum vulgare L.) is a long-day plant whose flowering is enhanced when the photoperiod is supplemented with far-red light, and this promotion is mediated by phytochrome. A chemically mutagenized dwarf cultivar of barley was selected for early flowering time (barley maturity daylength response [BMDR]-1) and was made isogenic with the cultivar Shabet (BMDR-8) by backcrossing. BMDR-1 was found to contain higher levels of both phytochrome A and phytochrome B in the dark on immunoblots with monoclonal antibodies from oat (Avena sativa L.) that are specific to different members of the phytochrome gene family. Phytochrome A was light labile in both BMDR-1 and BMDR-8, decreasing to very low levels after 4 d of growth in the light. Phytochrome B was light stable in BMDR-8, being equal in both light and darkness. However, phytochrome B became light labile in BMDR-1 and this destabilization of phytochrome B appeared to make BMDR-1 insensitive to photoperiod. In addition, both the mutant and the wild type lacked any significant promotion of flowering in response to a pulse of far-red light given at the end of day, and the end-of-day, far-red inhibition of tillering is normal in both, suggesting that phytochrome B is not involved with these responses in barley.     

Photoperiodism and photocontrol of stem elongation in two photomorphogenic mutants of Pisum sativum L.

The photomorphogenic mutation lv in the garden pea (Pisum sativum L.), which appears to reduce the response to light-stable phytochrome, has been isolated on a tall, late photoperiodic genetic background and its effects further characterised. Plants possessing lv have a reduced flowering response to photoperiod relative to wild-type plants, indicating that light-stable phytochrome may have a flower-inhibitory role in the flowering response of long-day plants to photoperiod. In general, lv plants are longer and have reduced leaf development relative to Lv plants. These differences are maximised under continuous light from fluorescent lamps (containing negligible far-red (FR) light), and decrease with addition of FR to the incident light. Enrichment of white light from fluorescent lamps with FR promotes stem elongation in the wild type but causes a reduction in elongation in the lv mutant. This “negative” shade-avoidance response appears to be the consequence of a strong inhibitory effect of light rich in FR, revealed in lv plants in the absence of a normal response to red (R) light. These results indicate that the wild-type response to the R: FR ratio may be comprised of two distinct photoresponses, one in which FR supplementation promotes elongation by reducing the inhibitory effect of R, and the other in which light rich in FR actively inhibits elongation. This hypothesis is discussed in relation to functional differentiation of phytochrome types in the light-grown plant. Gene lw has been reported previously to reduce internode length and the response to gibberellin A₁, and to delay flowering. The present study shows that the lw mutation confers an increased response to photoperiod. In all these responses the lw phenotype is superficially “opposite” to the lv phenotype. The possibility that the mutation might primarily affect light perception was therefore considered. The degree of dwarfing of lw plants was found to depend upon light quality and quantity. Dwarfing is more extreme in plants grown under continuous R light than in those grown in continuous FR or blue light or in darkness. Studies of the fluence-rate response show that the lw mutation imparts a lower fluence requirement for inhibition of elongation by white light from fluorescent lamps. Dark-grown lw plants are more strongly inhibited by a R pulse than are wild-type plants but, as in the wild type, this inhibition remains reversible by FR. Light-grown lw plants show an exaggerated elongation response to end-of-day FR light. Taken together, these findings indicate that the lw mutant may be hypersensitive to phytochrome action.     

Phytochrome Control of Chlorophyll Content in Mature Attached Leaves of Petunia axillaris

Petunia axillaris plants were grown under white light (photoperiod10 h). Pulses of end-of-day far-red light, but not red lightat the end of the day slowed down the accumulation of chlorophyllin expanding, greening leaves and reduced chlorophyll contentin mature leaves. The chlorophyll a / b ratio was unaffected.Low phytochrome photoequilibria reduced chlorophyll contentin mature leaves without affecting leaf area or d. wt. Thiseffect occurred even before senescence was triggered in thecontrol plants having high phytochrome photoequilibria     

A Temporarily Red Light-Insensitive Mutant of Tomato Lacks a Light-Stable,B-Like Phytochrome

We have selected four recessive mutants in tomato (Lycopersicon esculentum Mill.) that, under continuous red light (R), have long hypocotyls and small cotyledons compared to wild type (WT), a phenotype typical of phytochrome B (phyB) mutants of other species. These mutants, which are allelic, are only insensitive to R during the first 2 days upon transition from darkness to R, and therefore we propose the gene symbol tri (temporarily red light insensitive). White light-grown mutant plants have a more elongated growth habit than that of the WT. An immunochemically and spectrophotometrically detectable phyB-like polypeptide detectable in the WT is absent or below detection limits in the tri1 mutant. In contrast to the absence of an elongation growth response to far-red light (FR) given at the end of the daily photoperiod (EODFR) in all phyB-deficient mutants so far characterized, the tri1 mutant responds to EODFR treatment. The tri1 mutant also shows a strong response to supplementary daytime far-red light. We propose that the phyB-like phytochrome deficient in the tri mutants plays a major role during de-etiolation and that other light-stable phytochromes can regulate the EODFR and shade-avoidance responses in tomato.     

Phytochrome Regulation of the Response to Exogenous Gibberellins by Epicotyls of Vigna sinensis

The elongation rate of cowpea epicotyls from whole cowpea (Vigna sinensis) seedlings and derooted and debladed plants (explants) increased after the main light period (8-hour duration) was extended with either continuous low intensity tungsten light or brief (5 minutes) far-red (FR) irradiation. This end-of-day FR effect was reversed by red (R) irradiation suggesting the involvement of phytochrome. These results confirm and extend those obtained previously with other species. Localization studies indicate the epicotyl to be the site of the photoreceptor. Treatment of cowpea seedlings with paclobutrazol, a gibberellin (GA) biosynthetic inhibitor, abolished the FR promoted epicotyl elongation, indicating a role for GAs in this process. There was no significant difference in epicotyl elongation rates of R plus FR irradiated explants treated with GA₁ or GA₂₀ and R irradiated explants treated with GA₁. However, R irradiation inhibited subsequent epicotyl elongation of GA₂₀ treated explants. Moreover, the observation, using GC-MS, that GA₁ and GA₂₀ are native GAs in cowpea lends support to the concept that phytochrome may control the conversion of endogenous GA₂₀ to GA₁ in cowpea.     

Phytochrome A affects stem growth, anthocyanin synthesis, sucrose-phosphate-synthase activity and neighbour detection in sunlight-grown potato

Phytochrome action in fully de-etiolated sunlight-grown potato (Solanum tuberosum L.) was studied by comparing wild-type (WT) plants and transgenic plants with either a sense or an anti-sense phytochrome A (phyA) construction. Radial stem growth, anthocyanin levels, and sucrose-phosphate-synthase activity were directly related to the levels of phyA (severely reduced in transgenics with anti-sense phyA, normal in WT and increased in transgenic with sense phyA). In contrast, longitudinal stem growth was inversely related to the levels of phyA. Phytochrome A influenced stem-extension growth responses to red/far-red ratios perceived by stable phytochrome[s]. First, far-red light reflected by non-shading neighbours promoted stem growth in WT plants but transgenic plants with either increased or reduced phyA levels failed to respond to this light signal. Second, plants with low phyA levels also showed impaired sensitivity to reductions in end-of-day red/far-red ratios. In addition, phyA appears to perceive changes in irradiance reaching the stem: lowering the amount of red plus far-red light reaching the stem promoted stem growth in WT plants. This effect was exaggerated in phyA overexpressors and absent in phyA underexpressors. Thus, phyA is active in fully de-etiolated, sunlight-grown plants. Received: 4 October 1997 / Accepted: 24 October 1997     

Phytochrome Responses to End-of-Day Irradiations in Light-grown Corn Grown in the Presence and Absence of Sandoz 9789

Corn seedlings were grown in white light in the absence and presence of the chlorosis-inducing herbicide San 9789. The resulting green and achlorophyllous seedlings were used to investigate phytochrome-mediated responses to end-of-day far red irradiation and reversal of these responses by subsequent red irradiation. Mesocotyl and coleoptile elongation increased in response to end-of-day far red irradiation, whereas the anthocyanin content of the coleoptiles was decreased. All three responses were reversible by red irradiation following the far red. Dose-response curves for far red induction and red reversal of these responses did not differ significantly for plants grown in the presence or absence of San 9789. Thus, San 9789 appears to affect neither phytochrome itself nor the response system involved. Chlorophyll screening likewise does not affect phytochrome relationships for these responses.     

Ectopic expression of oat phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimatization

Survival of temperate-zone tree species under the normal summer-winter cycle is dependent on proper timing of apical growth cessation and cold acclimatization. This timing is primarily based on the perception of daylength, and through evolution many tree species have developed photoperiodic ecotypes which are closely adapted to the local light conditions. The longest photoperiod inducing growth cessation, the critical photoperiod, is inherited as a quantitative character. The phytochrome pigment family is the probable receptor of daylength, but the exact role of phytochrome and the physiological basis for the different responses between photoperiodic ecotypes are not known. This report shows for the first time that over-expression of the oat phytochrome A gene ( PHYA ) in a tree significantly changes the critical daylength and effectively prevents cold acclimatization. While the critical daylength for elongation growth in the wild-type of hybrid aspen ( Populus tremula × tremuloides ) was approximately 15 h, transgenic lines with a strong expression of the oat PHYA gene did not stop growing even under a photoperiod of 6 h. Quantitative analysis of gibberellins (GA) as well as indole-3-acetic acid (IAA) revealed that levels of these were not down-regulated under short days in the transgenic plants expressing high levels of oat PHYA , as in the wild-type. These results indicate that photoperiodic responses in trees might be regulated by the amount of PHYA gene expressed in the plants, and that the amount of phytochrome A (phyA) affects the metabolism of GAs and IAA.     

Is the far-red-absorbing form of Avena phytochrome A that is present at the end of the day able to sustain stem-growth inhibition during the night in transgenic tobacco and tomato seedlings?

Avena phytochrome A (phyA) overexpressed in tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon sculentum Mill) was functionally characterised by comparing wild-type (WT) and transgenic seedlings. Different proportions of phytochrome in its far-red-absorbing form (Pfr/P) were provided by end-of-day (EOD) light pulses. Stem-length responses occurred largely in the range of low Pfr/P (3–61%) for WT seedlings and in the range of high Pfr/P (61–87%) for transgenic seedlings. A similar shift was observed when the photoperiod was interrupted by short light pulses providing different Pfr/P ratios and followed by 1 h dark incubation. In other experiments, Avena phyA was allowed to re-accumulate in darkness and subsequently phototransformed to Pfr but no extra inhibition of stem extension growth was observed. In transgenic tomato seedlings the response to EOD far-red light was faster and the response to a far-red light pulse delayed into darkness was larger than in the WT. Avena phyA Pfr remaining at the end of the photoperiod appears intrinsically unable to sustain growth inhibition in subsequent darkness. Avena phyA modifies the sensitivity and the kinetics of EOD responses mediated by native phytochrome.Abbreviations EOD end-of-day - FR far-red light - Pfr/P pro-portion of phytochrome in its FR-absorbing form - phyA phyto-chrome A - phyB phytochrome B - R red light - RFR R to FR ratio - WT wild type We thank Dr Brian Thomas for providing the antibodies used in this work, and Federico Guerendiain for his excellent technical assistance. This work was financially supported by grants UBA AG 040 and Fundacion Antorchas A-12830/1-19 (both to J.J.C.), PID-CONICET (to R.A.S. and J.J.C.), United States Department of Energy DE-FG02-88ER13968 (to R.D.V.).     

Phytochrome and internode elongation in Chenopodium polyspermum L. sites of photoreception

The elongation of the fourth internode of fully green Chenopodium polyspermum L. is modulated by far-red light (FR) given in addition to the main light period. Two different types of organs are responsible for the photoreception of FR producing the end-of-day effect; the stem and the leaves situated just above and below the reacting internode. Photoreversibility can be obtained within certain limits in the two organs. Evidence is presented which shows that in the fully green plant there is an interorgan reaction whose primary reaction is the photoconversion of phytochrome.Abbreviations and Symbols D darkness - FR far red light - R red light - P phytochrome - P_FR phytochrome in the FR absorbing form - 9+15 D (or light treatment) photoperiod of a 9 h main light period followed by 15 h of D (or light treatment)