Correlation between far-red absorbing phytochrome and response in phytochrome-mediated anthocyanin synthesis

Abstract Mustard seedlings were light treated at 24 h after sowing (25°C) to induce phytochrome-mediated anthocyanin synthesis in cotyledons and hypocotylar hook. All light treatments were performed within the range of the reciprocity law. The in situ photoconversion kinetics of phytochrome (P_r→ P_fr) were measured under the same light treatment. It was found that between 0.4 and 1.0 relative P_fr level the amount of anthocyanin extracted from the organs at 52 h after sowing was linearily correlated with the amount of P_fr produced at 24 h in cotyledons and hypocotylar hook. It is concluded that an explanation of the fluence response function for red light mediated anthocyanin synthesis in the mustard seedling does not require the concept of active vs. bulk phytochrome.     

Regulation of enzyme levels by phytochrome in mustard cotyledons: Multiple mechanisms?

Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of P_fr, e.g. due to the liberation of a second messenger. We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL)⁺ and ribulosebisphosphate carboxylase (Carboxylase)⁺. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25° C). The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of P_fr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m^-2 red light and 67.5 mW m^-2 red light lead to the same time course), PAL induction is a graded response over a wide range of P_fr doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome.The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (P_fr). It seems that there is no feedback from pattern realization to pattern specification.Abbreviations P_fr the far-red absorbing, physiologically active form of phytochrome - P_r the red absorbing physiologically inactive form of phytochrome - P_total [P_r]+[P_fr] - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Carboxylase ribulosebisphosphate carboxylase (EC 4.1.1.39)     

Temporal Pattern of Gene Expression in Cotyledons of Mustard (Sinapis alba L.) Seedlings

The time course of appearance of competence to phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated accumulation of mRNAs encoding for SSU, CAB and the 23 kDa protein of the oxygen evolving complex of photosystem II (OEC). For each gene family a specific starting point of P_fr-induced mRNA accumulation was observed (SSU: 42 h; CAB: 36 h; OEC: 30 h). An increase of SSU-mRNA levels can be detected 24 h after sowing in dark-grown seedlings whereas for OEC the time points for the increase of mRNA are the same whether the seedlings are kept in darkness or induced by light via P_fr. For all gene families a responsiveness to P_fr (coupling point) could be demonstrated before the starting points. The coupling points are also gene specific (SSU: ca. 12 h; CAB and 23 kDa peptide of OEC: ca. 24 h). The responsiveness to light before the starting point indicates that the light-induced signal must be stored.     

Pattern formation underlying phytochrome-mediated anthocyanin synthesis in the cotyledons ofSinapis alba L.

The ability to respond to phytochrome (P_fr, the far-red light absorbing from of phytochrome) with anthocyanin synthesis appears first in some marginal regions of the abaxial epidermis of the mustard cotyledons and from there spreads gradually over the entire tissue (transient phase). The pertinent pattern is independent of environmental influences such as light quality and nutritional culture conditions. The competence for P_fr in the epidermal cells, with regard to the initial action of P_fr (concerning anthocyanin synthesis), appears considerably earlier than the ability for actual anthocyanin synthesis. An electron microscopical study of the ultrastructural changes occurring in vacuoles and plastids of the epidermal cells during the transient phase showed that a correlation only exists between the differentiation of central cell vacuoles, originating from the aleurone vacuoles, and the appearance of the ability to accumulate anthocyanin. It is suggested that the formation of a central cell vacuole is a prerequisite for anthocyanin accumulation in the epidermal cells of the mustard seedling cotyledons.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome - HS Hoagland's nutrient solution     

The effect of light pretreatments on phytochrome-mediated induction of anthocyanin and of phenylalanine ammonia-lyase

Induction by light of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and of anthocyanin in cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by a light pretreatment which operates through phytochrome. If PAL or anthocyanin is induced by a light pulse, the effectiveness of phytochrome (P_fr) is strongly increased by a light pretreatment; however, if the increase of the PAL level or synthesis of anthocyanin is elicited by continuous far-red light (operating via phytochrome in the High Irradiance Response), effectiveness of light is strongly reduced by the same light pretreatment. This reduction of effectiveness is correlated with a decrease of total phytochrome (P_tot) caused by the light pretreatment. It is argued that the observations are compatible only with the open phytochrome-receptor model as suggested by Schäfer (J. Mathem. Biol. 2, 41–56, 1975). The peaks of the time courses of the PAL levels under continous far-red light are located at 48 h after sowing and do not depend on the original level of phytochrome. The decrease of the PAL levels beyond 48 h after sowing takes place independently of phytochrome and of the actual level of PAL.Abbreviations P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - P_tot total phytochrome (P_r+P_fr) - {ie369-1} [P_fr] /[P_tot], photoequilibrium of phytochrome at wavelength - HIR High Irradiance Response - PAL phenylalanine ammonialyase (EC 4.3.1.5)     

Timing of the initial action of phytochrome with regard to protochlorophyll synthesis in the mustard seedling

Summary Significant accumulation of photoconvertible protochlorophyll(ide) in the cotyledons of the mustard seedling takes place from 24 h after sowing onwards (25° C). The rate of accumulation in darkness is greatly increased by a pretreatment with red or far-red light. The strong effect of continuous red light, given from the time of sowing, remains fully reversible by a 756 nm-light pulse up to about 18 h after sowing. On the other hand, the effect of continuous far-red light which can be detected at 15 h after sowing is not influenced by a subsequent application of 756 nm-light pulses. An interpretation of the data requires the concept that continuous red light and continuous far-red light act from different sites. This conclusion is based on a comparison of the present data with the earlier published data on phytochromemediated anthocyanin synthesis in the mustard seedling cotyledons.Abbreviations PChl protochlorophyll(ide) - Chl chlorophyll(ide) - P_tr far-red absorbing form of the phytochrome system (physiologically active) - P_r red absorbing form of the phytochrome system - [P_tot] [P_r]+[P_fr] Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 46).     

Spectrophotometric characterization of phytochromes in dimorphic cocklebur seeds in relation to capacity for germination

Phytochrome was measured spectrophotometrically in different tissues of the upper (positively photoblastic) and lower (negatively photoblastic) seeds of the cocklebur (Xanthium pennsylvanicum Wallr.). Axial parts of the seeds, in particular parts of the radicle, contained high levels of phytochrome, while cotyledonary parts contained only low levels. These results were consistent with the distribution of the light-sensitive areas of the seeds that were associated with germination. Phytochrome levels in both types of dimorphic seeds increased gradually with increasing duration of dark imbibition for 4–8 h, then the rates of increase in levels of phytochrome accelerated. In both types of seed, some phytochrome was measurable even before imbibition. In the lower seeds, up to 20% of the phytochrome was occasionally observed as P_fr in samples imbibed in darkness for a short time (up to 12 h). A slight blue shift of the peak of P_T in the difference spectrum of phytochrome was observed in the case of lower seeds imbibed for 0–2 h. These results suggest that, to some extent, the lower axes contain dehydrated P_fr or intermediate(s) in the photoconversion of phytochrome. The dark reactions of P_fr were also examined in excised axes of both types of dimorphic seed after they had been pre-imbibed for 16 h in darkness. Dark destruction of P_fr was observed in both types of seed. In addition, net increases in levels of P_r were observed in the dark controls and in the samples irradiated with red light after the level of P_fr diminished. No ‘inverse’ dark reversion from P_r to P_fr was detected. Thus, after 16 h of imbibition, there were no differences in terms of properties of phytochrome between the two types of seed, and the different responses to light of upper and lower seeds might depend mainly on a difference in the physiological state of the two types of seed rather than the properties of phytochrome.     

Light requirement,phytochrome and photoperiodic induction of flowering of Pharbits nil Chois

The low chlorophyll content of cotyledons of Pharbitis nil grown for 24 h in far-red light (FR) or at 18° C in white light from fluorescent lamps (WL) allows spectrophotometric measurement of phytochrome in these tissues. The (A) measurements utilize measuring beams at 730/802 nm and an actinic irradiation in excess of 90 s. The constancy of the relationship between phytochrome content and sample thickness confirms that, under these conditions of measurement, a true maximum phytochrome signal was obtained. These techniques have been used to follow changes in the form and amount of phytochrome during an inductive dark period for flowering. Following exposure to 24h WL at 18° C with a terminal 10 min red (R), P_fr was lost rapidly in darkness and approached zero in less than 1 h; during this period there was no change in the total phytochrome signal. Following exposure to 24 h FR with a terminal 10 min R, P_fr approached zero in 3 h, and the total phytochrome signal decreased by about half. The relevance of these changes to photoperiodic time measurement is discussed.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Destruction and possible de novo synthesis of phytochrome in subcellular fractions of laminae from Avena sativa L.

Phytochrome was determined in etiolated laminae of Avena sativaL. either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0–24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reacheda minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids.Abbreviations P_tot total phytochrome - P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - ER endoplasmic reticulum     

Coaction of three factors controlling chlorophyll and anthocyanin synthesis

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, P _fr) and an excision factor. It was found that the three factors operate additively provided that the P _fr level is high enough. When the P _fr level is below approximately 1 per cent (<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low P _fr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.Abbreviations Chl chlorophyll(ide) a - P _fr far-red absorbing form of phytochrome - P _fr/P _tot ratio at photoequilibrium - RL red light - FR far-red light - GL green light - RG9 light long wavelength far-red light - WL white light     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

Investigations on the role of ethylene in phytochrome-mediated photomorphogenesis

The etiolating, intact mustard (Sinapis alba L.) seedling exhibits a distinct temporal pattern of ethylene production. Light, operating through phytochrome, increases the rate of ethylene production without changing the pattern. Ethylene production of the isolated plant parts (segments), added together, exceed the production of the intact system even if the wound effect is taken into account. There is no significant light effect on ethylene production of the segments. Phytochrome-mediated anthocyanin synthesis in the cotyledons is inhibited by ethylene. The responsiveness towards ethylene of the anthocyanin producing metabolic chain is decreased by phytochrome. As anthocyanin synthesis is only partly inhibited under saturating ethylene concentrations in the atmosphere around the seedlings (100 l l^–1), a twofactor analysis becomes feasible. This analysis leads to the result that phytochrome and ethylene show multiplicative behavior, meaning that phytochrome and ethylene act on the same metabolic sequence (leading to anthocyanin) but independently of each other, and at different sites. Therefore, the hypothesis that ethylene mediates the action of phytochrome in anthocyanin synthesis and photomorphogenesis in general appears to be inapplicable.Abbreviations P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome, i.e. [P_r]+[P_fr]     

Control by light of hypocotyl growth in de-etiolated mustard seedlings

After sowing, mustard (Sinapis alba L.) seedlings were grown for 48 h in white light (25°C). These fully de-etiolated, green seedlings were used as experimental material between 48 and 72 (84) h after sowing. The question researched was to what extent control by light of hypocotyl elongation is due to phytochrome in these seedlings. It was found that the light effect on hypocotyl growth is very probably exerted through phytochrome only. In particular, we found no indication for the involvement of a specific blue light photoreceptor pigment.Abbreviations HIR high irradiance reaction - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - Pot total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_tot] - red red light - fr far-red light - wl white light - bl blue light - di dichromatic irradiation - l hypocotyl length     

Photocontrol of anthocyanin formation in turnip seedlings

Summary As measured by in vivo spectrophotometry the phytochrome content in etiolated turnip seedlings was higher in cotyledons than in hypocotyls; in the latter, it is confined to the apical part. During early growth in darkness the amount increased in both tissues to a maximum, reached about 40 hours after sowing; the levels then gradually declined. Separation of seedlings into hypocotyl and cotyledons increased the rate of phytochrome loss in the former, but not in the latter.Following 5 minutes of red light P _frdecayed very rapidly in darkness; after 1.5 hours all of the phytochrome was present as P _r, which was presumably not converted initially. In continuous red light the total phytochrome was reduced to below the detection level within 3 hours. Seedling age markedly affected the loss of phytochrome following red light; more was destroyed in older than in younger hypocotyls and apparent new synthesis occurred only in young seedlings. The capacity to synthesise phytochrome differed in cotyledons and hypocotyl. In cotyledons, synthesis occurred following shots of red light varying from 10 seconds, to 6×I minute, but the amount of newly formed phytochrome was not related to the amount destroyed: after 5 hours of continuous red light no new synthesis occurred. In hypocotyls, the amount of phytochrome synthesised was related to the amount previously destroyed, and the phytochrome content after 24 hours of darkness was similar following all red light treatments of 1 minute or longer: new synthesis occurred following 5 hours of continuous red light.In far-red light phytochrome decayed very slowly, approaching the limit of detection after 48 hours. In cotyledons some loss was already observed after 5 hours of far-red and, in hypocotyls, after about 10 hours.These results are discussed in relation to the possible role of phytochrome as the pigment mediating anthocyanin synthesis in prolonged far-red light.     

Properties of phytochrome in gymnosperms

Summary Addition of water to dry seeds of Pinus spp. increased the detectable phytochrome immediately and the level reached after 2 h in darkness was retained for at least 20 h at 20° C. The in-vivo difference spectra of phytochrome in Pinus seeds showed absorption maxima at approximately 656 nm and at 710 nm to 715 nm. An isosbestic point was observed at about 680 nm. Shifts towards longer wavebands were obtained especially with tissue containing substantial amounts of chlorophyll and are, therefore, not due to diverse types of phytochrome. Embryo tissue of Ginkgo biloba showed also a maximum in R at 655 nm but the peak in FR occurred at a longer wavelength, 725 nm. This was confirmed by determining action spectra for the phototransformations P_rP_fr.The dark reactions of phytochrome in Pinus differed from those in Ginkgo. Following a short exposure to R light, the total quantity of photoreversible pigment in Pinus seeds remained constant for several hours in darkness at room temperature. Dark reversion of P_fr occurred extremely rapidly and tP_fr 50 was only 0.3 h. In Ginkgo embryos total phytochrome in darkness following a brief exposure to R light was not completely stable. Reversion of P_fr was much slower and tP_fr 50 was slightly less than 2 h.It is concluded that, at least as regards the spectral qualities, the phytochrome in Gymnospermae differs from that of Angiospermae and is apparently also not identical in Coniferae and Ginkgoinae. Abbreviations. R = red; FR = far-red; R/FR ratio = (A) red max./(A) far-red max. of difference spectrum. The peak positions and the isosbestic point are estimated from the difference spectra and are approximate only. P_r = red-absorbing form of phytochrome, P_fr = far-red absorbing formThis work was carried out with financial support from the Netherlands Organisation for Pure Scientific Research (Z.W.O.).312th Communication.     

Photocontrol of stem elongation in light-grown plants of Fuchsia hybrida

Stems of the caulescent long-day plant, Fuchsia hybrida cv Lord Byron, showed 2 types of response to light. In one, internode length was increased by far-red irradiation given at the end of an 8 h photoperiod: the response was no greater with prolonged exposure and was less when the start of far-red was delayed. The effect of far-red was reversible by a subsequent exposure to red light. Internode length was inversely proportional to the P_fr/P ratio established before entry to darkness and there was no evidence for loss of P_fr during a 16 h dark period. The inhibitory effect of P_fr acted at a relatively late stage of internode growth. With the development of successive internodes a second response appeared in which stems lengthened following prolonged daily exposures to red or far-red light, or mixtures of the two, or to brief breaks with red or white light. In these later internodes, a short exposure to far-red near the middle of the night was not reversible by red because red alone promoted elongation at this time. Internode length increased with increase in the daily duration of light and, when light was given throughout an otherwise dark period of 16 h, with increase in illuminance to a saturation value of 200 lx from tungsten lamps. Elongation increased as a linear function of decrease in photostationary state of phytochrome down to P_fr/P0.3; however, internodes were shorter in far-red light than in 25% red/red+far-red. It was concluded that stem length is a net response to two modes of phytochrome action. An inductive effect of P_fr inhibits a late stage in internode expansion, and a phytochrome reaction which operates only in light (and may involve pigment cycling) promotes an early stage of internode development. Stem elongation is thus a function both of the daily duration of light and its red/red+far-red content. The outgrowth of axillary buds was controlled by the first type of phytochrome action only.Abbreviations and symbols FR far red light - R red light - P phytochrome - P_fr phytochrome in the far-red light absorbing form - SD 8 h short days - LDP long-day plant - SDP short-day plant     

Time-dependent changes in the responsiveness to light of phytochrome-mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard ( Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.     

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.