Polarotropism and Photomovement of Chloroplasts in the Protonemata of the Ferns Pteris and Adiantum: Evidence for the Possible Lack of Dichroic Phytochrome in Pteris

The actions of red and blue light in the photomovement of chloroplastsand the polarotropic response were studied in the protonemataof the homosporous ferns Pteris vittata L. and Adiantum capillus-venerisL. In Pteris, polarotropism could be induced with blue lightbut not with red light, while both colors of light were effectivein Adiantum protonemata. The photomovement of chloroplasts inthe two species studied by both polarized light and microbeamirradiation, also revealed similar responses to red and bluelight as the polarotropism; i.e. both colors of light were effectivein Adiantum but only blue light was active in Pteris. The resultsin Adiantum were consistent with previous results, which ledto the conclusion that both phytochrome and a blue light-absorbingpigment are involved in the two responses (Kadota et al. 1982,1984, Hayami et al. 1986, Yatsuhashi et al. 1985). By contrast,phytochrome is not involved in either polarotropism or chloroplastmovement in Pteris. Since the phytochrome system is evidentlyactive in every other photoresponses so far investigated inPteris as well as in Adiantum, the present study suggests thata phytochrome system specific to polarotropism and to photomovementof chloroplasts is absent in Pteris. Discussions are presentedon the possible involvement of two phytochrome populations ina fern gametophyte cell and on the possible lack of dichroicphytochrome in Pteris. (Received October 7, 1988; Accepted March 8, 1989)     

Phytochrome in the Fern, Adiantum capillus-veneris L.: Spectrophotometric Detection in Vivo and Partial Purification

Spectrophotometric studies of fern phytochrome were performedusing dark-grown leaves of Adiantum. The absorbance differencespectrum between the red- and far-red-light irradiated sampleshowed a photoreversible absorbance change in the far-red region,with a maximum located at 728–730 nm. The concentrationof phytochrome was highest at the leaf tips and decreased graduallyalong the leaf axis. As in the case of angiosperm phytochrome,the level of fern phytochrome decreased under continuous whitelight, and the level increased again when deetiolated tissuewas transferred back to the dark. When the fern tissue was exposedto a pulse of red light, the dark reversion of P_FR to P_R tookplace with almost no destruction of P_FR. Phytochrome could beextracted from light-grown young leaves of the fern with a slightlyalkaline, aqueous buffer that contained 1 M NaCl. The differencespectrum of the partially purified phytochrome from fern wassimilar to that of partially degraded phytochrome from angio-sperms.A polyclonal antibody raised against phytochrome from etiolatedrye seedlings immuno-stained (albeit weakly) a 110-kDa polypeptideafter fractionation by SDS-polyacrylamide gel electrophoresisof the preparation of fern phytochrome. The band was very probablyfern phytochrome since it emitted zinc-induced fluorescence. (Received July 12, 1990; Accepted October 5, 1990)     

Phytochrome Activation of K+ Channels and Chloroplast Rotation in Mougeotia: the Escape Times

Red light mediates chloroplast movement and increased activityof calcium-activated potassium channels on the plasma membraneof the alga Mougeotia sp. (UTEX LB 734). When activation ismediated by phytochrome, a far-red light irradiation given sometime after the red light irradiation will reverse the effectof the red light, due to phytochrome photoreversibility. Wecharacterized the escape times (time required for loss of photoreversibility)for these two processes to compare the transduction pathwaysinvolved in chloroplast rotation and channel activation. Theescape time for chloroplast rotation was 2.5 min after red lightirradiation (red and far-red light irradiations were 30 s).For channel activation, shorter red and far-red light irradiations(10 s) had to be used to obtain an escape time of 20 s. Thedifference in the escape times suggests that there is relativelyrapid divergence in the transduction pathways leading from phytochromeactivation (only one molecular species of phytochrome is foundin Mougeotia) to each of the two responses in the same cellularsystem. Because channel activation occurs 2–4 min afterirradiation while the escape time is 20 s, it is unlikely thatphytochrome acts directly on the channel. (Received September 26, 1995; Accepted December 28, 1995)     

A Comparison of Seed and Seedling Phytochrome in Avena sativa L. using Monoclonal Antibodies

Hilton, J.R. and Thomas, B. 1985. A comparison of seed and seedlingphytochrome in Avena saliva L.using monoclonal antibodies.—J.exp. Bot. 36: 1937–1946.The kinetics of phytochrome accumulationduring imbibition and germination of seeds of Avena saliva L.cv. Saladin has been determined in vivo by spectrophotometryand in extracts by using Enzyme–Linked Immunosorbent Assay(EL1SA). In vivo measurements show two phases of phytochromeincrease. The first occurs during the initial 2 h of imbibitionand is associated with the hydration of the seed proteins; thesecond larger increase begins after 16 h, due probably to denovo synthesis. An ELISA using monoclonal antibodies purifiedfrom dark grown Avena seedlings detected only the second increasein phytochrome content. Mixing experiments indicate that theinability to detect phytochrome by ELISA during the first 16h is not due to the presence of inhibitors in the extracts.It is concluded that pre–existent seed phytochrome isantigenically dissimilar to seedling phytochrome. These twopools of phytochrome are stable and unstable respectively withregard to Pfr destruction. Key words: Immunology, phytochrome, seed     

Dichroic Orientation of Phytochrome Intermediates in the Pathway from PR to PFR as Analyzed by Double Laser Flash Irradiations in Polarotropism of Adiantum Protonemata

The polarotropic response in protonemata of the fern Adiantumis regulated by phytochrome (Kadota et al. 1984); P_R and P_FRhave been shown to be dichroically oriented parallel and normalto the cell surface, respectively (Kadota et al. 1982). Thischange in the dichroic orientation of phytochrome during photoconversionwas analyzed by a newly-built, polarization plane-rotatabledouble laser flash irradiator. A polarotropic response was effectivelyinduced with a flash of polarized red (640 nm) light (6xl0^–7s) having the vibration plane of the electrical vector parallelto the protonemal cell axis. When a flash of polarized far-red(710 nm) light (6xl0^–7s) was given 30 sec after the redflash, the red flash-induced response was reversed by a far-redflash vibrating normal to the cell axis but not by one vibratingparallel. However, when given 2 µs or 2 ms after the redflash, the polarotropic response was not reversed by a polarizedfar-red flash vibrating normal to the cell axis but was reversedby a parallel-vibrating flash. These results suggest that theorientation of phototransformation intermediates existing 2µs or 2 ms after a red flash is still parallel to thecell surface, and that the change in the orientation of phytochromemolecules occurs between 2 ms and 30 s after the red flash. (Received February 3, 1986; Accepted April 23, 1986)     

The Effect of Gabaculine on Germination and Gametophyte Morphogenesis of Anemia phyllitidis L.Sw.

Gabaculine, applied to germinating spores of the fern Anemiaphyllitidis in concentrations between 10^–4 M and 10^–6M inhibits chlorophyll synthesis together with considerablesimplification of chloroplast ultrastructure. Though gabaculinereduced phytochrome-induced morphogenetic effects (cell shape,inhibition of hormonal antheridium induction), the rate of redlight-induced spore germination, however, has not been affectedby the inhibitor. This indicates significant differences betweenthe two types of photomorphogenetic reactions regarding theirdependency on phytochrome homeostasis. (Received April 21, 1986; Accepted October 22, 1986)     

Photoinduction of Spore Germination in a Fern, Mohria caffrorum

Irradiation of spores of the fern Mohria caffrorum Sw. witheither red light (67.4 µW cm^–2) or far-red light(63.2 µW cm^–2) for a period of 24 h induced maximumlevels of germination. Brief irradiations with blue light (127.6µW cm^–2) administered before or after photoinductioncompletely nullified the effects of red or far-red light; however,with prolonged exposure to blue light, germination levels roseto near maximum. The similar effects of red and far-red lightin promoting spore germination makes the involvement of phytochromein this process questionable. Based on energy requirements,the promotive and inhibitory phases of blue light appear toinvolve independent modes of action. Mohria caffrorum, ferns, spore germination, photoinduction, phytochrome     

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     

Particle-bound phytochrome: Differential pigment release by surfactants, ribonuclease and phospholipase C

Surfactants and hydrolytic enzymes were used to probe the natureof the constituents) to which phytochrome binds in paniculatefractions from red-irradiated Cucurbita. [¹⁴C]-choline and [³H]-uridinepre-labelled tissue was used to monitor the release of phospholipidsand RNA by these agents. Ribonuclease (RNase) digestion of 20,000xgpellets eliminates both the phytochrome and ribonucleoprotein(RNP) which cosediment at 31S. Little [¹⁴C]-choline occurs inthe 31S fraction and the amount is not changed by RNase digestion.This is further evidence that phytochrome binds directly tothe RNP in the 31S fraction rather than to any membranous materialpresent. The distribution profile of the RNA in a second ( =‘heavy’)phytochrome fraction does not correlate with that of the pigment.This suggests that the phytochrome in this fraction is not boundto RNP. The RNA is of ribosomal origin but much less degradedthan that of the 31S RNP and is resistant to RNase digestion.Phospholipase C releases>80% of the [¹⁴C]-choline from the‘heavy’ fraction without freeing phytochrome. Thisindicates that the pigment does not bind to the polar head groupsof the membrane phospholipids present. Low concentrations ofdeoxycholate dissociate phytochrome from this fraction withoutreleasing substantial quantities of integral membrane proteinsor phospholipids. Some RNP is dislodged by the surfactant butthe phytochrome and RNP are not released as a complex. The datasuggest that the pigment in the ‘heavy’ fractionmay be loosely bound to a protein constituent rather than toRNP or polar phospholipids. ¹This work was done while on sabbatical leave from the WeizmannInstitute of Science, Rehovot, Israel. (Received April 1, 1976; )     

Changes in Phytochrome Content during Imbibition in Spores of the Fern Lygodium japonicum

The phytochrome content was determined in intact fern sporesof Lygodium japonicum (Thunb.) Sw. by difference spectrophotometry.The spectral characteristics thus estimated in spores whichhad been imbibed for 9 days in darkness were: far-red maximumat 730?2.5 nm, red maximum at 662?1.5 nm and isosbestic pointat 684.5?1.4 nm. A detectable amount of phytochrome first appearedafter 3 days of dark imbibition, and the level then increasedduring the rest of the imbibition period. On the 7th day, thephytochrome content leveled off. During the dark imbibitionperiod, the phytochrome was revealed to be in the P_R form. (Received February 22, 1982; Accepted July 9, 1982)     

The Interactive Effects of Phytochrome, Nitrate and Thiourea on the Germination Response to Alternating Temperatures in Seeds of Ranunculus sceleratus L.: A Quantal Approach

Probert, R. J., Gajjar, K. H. and Haslam, I. K. 1987. The interactiveeffects of phytochrome, nitrate and thiourea on the germinationresponse to alternating temperatures in seeds of Ranunculussceleratus L.: A quantal approach.—J. exp. Bot. 38: 1012–1025. The interactive effects of phytochrome, potassium nitrate andthiourea on the germination response to alternating temperaturesin achenes (seeds) of Ranunculus sceleratus L. were studied.Using thermogradient bars, high levels of germination were recordedover a broad range of alternating temperatures providing seedsreceived daily irradiations. Reduced germination in temperaturecycles with a relatively long warm phase was related to thelevel of the active form of phytochrome (Pfr). Dose-responseexperiments to red light (R) and temperature shifts showed thatthe actions of Pfr and alternating temperatures were interdependent.Maximum germination was recorded when intermittent pulses ofR were combined with daily 4 h temperature shifts from 16°Cto 26°C. Whilst probit analysis showed that potassium nitrateand thiourea both increased population sensitivity to temperatureshifts, thiourea was a more potent stimulant. Although the effectof both chemicals was dependent on phytochrome photo-equilibriumthe threshold level of Pfr required for thiourea action wasclearly much lower than that required for nitrate action. Thioureapotentiated a response to daily temperature shifts even whenPfr was at a low, normally inhibitory level. These results indicatedifferent mechanisms of action for potassium nitrate and thioureain relation to phytochrome controlled seed germination. Key words: Phytochrome, nitrate, thiourea, alternating temperatures, germination     

Germination of seeds in Hyptis suaveolens Poit

Seeds of Hyptis suaveolens require long illumination periodsto promote full germination, both light and dark germinationbeing controlled by the phytochrome system. Germination in thisspecies is inhibited both by relatively low (up to 20°C)and high temperatures (45°C). Experiments in which seedswere transferred after different periods from sub-optimal orsupra-optimal temperatures to a favourable one, suggest thatboth high and low temperature inhibition may be explained onthe basis of phytochrome action. Thus, a temperature of 20°Cinduces dark dormancy due, probably, to dark reversion of phytochrometo the inactive form; whereas, at 45°C the dominant processseems to be phytochrome decay. No phytochrome loss has beenobserved at 10°C. If, however, phytochrome levels are reducedby a particular treatment, no irreversible alteration is produced;seeds simply require longer illumination periods under diesecircumstances. A low initial concentration of phytochrome couldaccount for the requirement of long illumination periods. Other factors affecting germination in this species, such asalternating temperatures, gibberellic acid and time of storage,are discussed. ¹ Present address: Instituto Venezolano de Investigaciones Cientificas,Apartado 1827, Caracas, Venezuela. (Received January 21, 1971; )     

The Deduced Amino Acid Sequence of Phytochrome from Adiantum Includes Consensus Motifs Present in Phytochrome B from Seed Plants

A cDNA for the phytochrome of the fern Adiantum capillus-venerisL. was cloned and sequenced. The deduced phytochrome is 50{smalltilde}55% identical to phytochromes of seed plants, and 68%identical to Selaginella phytochrome. Regions resemble thosein previously characterized phytochromes from ferns, lower plantsand seed plants. ³Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka,Tsukuba-shi, Ibaraki, 305 Japan ⁴Present address: Plant Growth Regulation Laboratory, The Instituteof Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,Saitama, 351-01 Japan ⁵Present address: Advanced Research Laboratory, Hitachi, Ltd.,Hatoyama, Saitama, 350-03 Japan     

Photoregulation of Phytochrome Synthesis in Germinating Embryos of Avena sativa L.

Hilton, J. R. and Thomas, B. 1987. Photoregulation of phytochromesynthesis in germinating embryos of Avena sativa L.—J.exp. Bot. 38: 1704–1712. The effect of light on the accumulation of phytochrome in germinatingAvena embryos was determined. A quantitative ELISA using monoclonalantibody AFRC MAC 56 was used to measure specifically type 1(or dark) phytochrome. A pulse of red light given after 14 himbibition but prior to the onset of type 1 phytochrome synthesis,strongly inhibited subsequent type 1 phytochrome accumulation.This effect of red light at 14 h was reversible by far-red lightindicating the involvement of phytochrome. Red light also inhibitedphytochrome synthesis after 18 h and 24 h imbibition but after24 h, far-red light did not reverse the effect. The effect ofred light treatment at 18 h was reversed by giving a pulse offar-red light at any time up to 30 h. Seed germination was notinfluenced by light under the conditions of these experiments.It is proposed that type 2 (or light) phytochrome may be responsiblefor photoregulation of type 1 phytochrome synthesis in germinatingAvena embryos. Key words: Photoregulation, phytochrome, seed.     

Photoregulation of Phenylalanine Ammonia Lyase is not Correlated with Anthocyanin Induction in Photomorphogenetic Mutants of Tomato(Lycopersicon esculentum)

Photoregulation of phenylalanine ammonia lyase (PAL)(EC 4.3.1.5 [EC] )was analyzed in wild type (WT) and mutants: phytochrome dencient-awrea(au), high pigment exhibiting exaggerated phytochrome response(hp) and the double mutant (au.hp) of tomato (Lycopersicon esculentum(Mill.) cv. Ailsa Craig). Red light, acting via phytochrome,stimulates PAL activity in cotyledons and hypocotyls of tomatoseedlings. The time course of photoinduction of PAL in cotyledonsof the mutants (au and au.hp) and WT seedlings has a peak ofactivity at 4 h, after which the activity falls sharply, exceptin hp seedlings where activity is maintained at a high level.In hypocotyls, photoinduction of PAL also shows an initial rise,reaching a maximum at 3 h, followed by a sharp decline in themutants (au and au.hp) and WT seedlings. However in hp seedlingsphotoinduction of PAL is about 3 fold that in WT. The photoinductionof PAL appears to be dependent on de novo synthesis of proteinand nucleic acids. The use of a PAL specific inhibitor a-aminooxyß-phenylpropionic acid indicated that PAL is an essentialcomponent of the anthocyanin biosyn-thetic pathway in the tomatoseedlings. However, a comparison of anthocyanin biosynthesis[Adamse et al. (1989) Photochem. Photobiol. 50: 107] and PALphotoinduction data revealed that phytochrome mediated inductionof PAL and anthocyanin in the tomato seedlings are not correlated.While au and au.hp mutant seedlings show a similar increasein PAL level as in the WT, there is little formation of anthocyaninin these mutant seedlings. The results indicate that, in contrastto the photoregulation of anthocyanin synthesis which is dependenton the presence of the labile phytochrome (IP) pool in tomatoseedlings, the photoinduction of PAL is mediated via a smallpool of phytochrome in au mutant: stable phytochrome (sP) ora residual /P pool. (Received August 6, 1991; Accepted September 27, 1991)     

Photoinhibition of Seed Germination in the Maritime Plant Matthiola tricuspidata

The three-horned stock, Matthiola tricuspidata (L.) R. Br. isa widespread annual plant of the Mediterranean sandy shores.Its seeds are dark germinating and negatively photosensitive,in accordance with our previous findings for a number of othermaritime plants. Full germination was obtained at a wide rangeof temperatures (5-25 °C) in the dark. Inhibition of germinationunder light of various spectral qualities could be generallycorrelated, negatively and positively, respectively, with phytochromephotostationary state (ø) and relative cycling rate ofphytochrome (H). The inhibition of germination by white (fluorescent),blue and far-red light, applied either continuously or intermittently,consistently showed a linear dependence upon the logarithm ofthe flux density of the irradiation. The resulting photoinhibitioncurves had parallel slopes and, compared to those of other maritimeplants, they were shifted to higher flux densities, Continuousblue or far-red irradiations, both establishing a similar øvalue (0·26), resulted in statistically similar regressioncurves, thus favouring the hypothesis that phytochrome is thesingle photoreceptor in the photoinhibition of seed germination.Copyright1994, 1999 Academic Press Matthiola tricuspidata (L.) R. Br., three-horned stock, seed germination, light, photoinhibition, phytochrome     

Effects of Blue Light Pretreatments on Internode Extension Growth in Mustard Seedlings after the Transition to Darkness: Analysis of the Interaction with Phytochrome

The effects of blue light (B) pretreatments on internode extensiongrowth and their possible interaction with phytochrome mediatedresponses were examined in Sinapis alba seedlings grown for11 d under 280 µmol m^–2 s^–1 of continuousblue-deficient light from low pressure sodium lamps (SOX). SupplementaryB (16 µmol m^–2 s^–1) caused no detectable inhibitionof the first internode growth rate under continuous SOX, butgrowth rate was inhibited after transfer to darkness. This effect,and the growth promotion caused by far-red bend-of-day' lightpulses were additive. The addition of B at 16 µmol m^–2s^–1 during 11 d, or only during the first 9 or 10 d orthe latest 0.75, 1 or 2 d of the SOX pretreatment caused approximatelythe same extent of inhibition after the transition to darkness.A single hour of supplementary B before darkness caused morethan 50% of the maximum inhibition. However, 24 h of lower fluencerates of B (4 or 7 µmol m^–2 s^–1) were ineffective.Covering the internode during the supplementary B period didnot prevent the response to B after the transition to darkness.Far-red light given simultaneously with B (instead of the SOXbackground) reduced the inhibitory effect of B. Above a given threshold fluence rate, B perceived mainly inthe leaves inhibits extension growth in subsequent darkness,provided that high phytochrome photo-equilibria are presentduring the irradiation with B. Once triggered, this effect doesnot interact significantly with the ‘end-of-day’phytochrome effect. Key words: Blue light, extension growth, phytochrome     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

Photomorphogenesis in Pteris vittata IV. Action spectra for inhibition of phytochrome-dependent spore germination

Action spectra between 350 and 500 nm for the inhibition ofphytochrome-dependent spore germination in the fern Pteris vittatawere obtained. Both action spectra obtained before and afterred light irradiation have peaks at about 440 nm and 380 nmand shoulders from 440 to 480 nm. These results suggest thatthe phytochrome system is not involved in the inhibitory processof spore germination induced by short irradiation with bluelight. (Received October 8, 1970; )     

Nature of 'Pollinator' Effect in Potato (Solanum tuberosum L.) Haploid Production

Haploids (2n =24) of the common tetraploid (2n=48) potato (SolanumtuberosumL.) provide promising material for attacking many problemsconcerned with the genetics, cytogenetics and breeding of thisspecies. Interspecific 4xx2xcrosses betweenSolanum tuberosumgp.Andigenaorgp.Tuberosumcultivars as pistillate parents andSolanum tuberosumgp.Phurejaassource of pollen (hereafter ‘pollinator’) have beenused to produce maternally derived haploids through parthenogenesis.This paper discusses the nature of the ‘pollinator’effect in haploid extraction. The ‘pollinator’ hada significant effect on haploid frequencies following 4xx2xcrosses.The ‘pollinator’ effect seems to operate via theendosperm, in which haploid (n=2x) embryos are associated withhexaploid endosperm. A superior ‘pollinator’ appearsto have its effect by contributing two haploid (n) gametes tothe central cell. 2n pollen; double fertilization; endosperm; ploidy manipulations; Solanum tuberosum