Differences in Photoresponse and Phytochrome Spectrophotometry Between Etiolated and De-etiolated Pea Stem Tissue

Morphologically similar pea plants having a 4-fold difference in spectrophoto-metrically detectable phytochrome can be produced by pretreatment of etiolated plants with red light (R) or with red and far-red light combined (RF). A search for response differences which could be ascribed to differences in phytochrome content has resulted only in the establishment of differences due to de-etiolation. Segments of etiolated plants differ from those of plants de-etiolated by R and RF pretreatments in 2 ways. Segments from etiolated plants appear to respond rapidly to the far-red absorbing form of phytochrome (P_FR), while segments from de-etiolated plants do not respond rapidly to P_FR. This statement is based upon 2 observations: (i) the red light induced growth inhibition in segments from etiolated plants rapidly escapes reversibility by far-red light, while with segments from R or RF pretreated plants, the red light effect is fully reversed by subsequent far-red light for up to 2 hr; and (ii) segments from etiolated plants were inhibited to a greater degree than were segments from RF pretreated plants when various photostationary state levels of P_FR were maintained for 30 or 90 min and then removed by photoconversion to P_R. The in vivo nonphotochemical transformation curves of the phytochrome of etiolated and RF pretreated plants appear to differ in 2 related respects: (i) the amount of phytochrome destroyed in de-etiolated tissue is greater than that in etiolated tissue, perhaps as a result of the fact that (ii) the rate and extent of apparent reversion of P_FR to P_R in etiolated tissue is about twice that in de-etiolated tissue.     

Response of tissue with different phytochrome contents to various initial photostationary States

Pretreatment of etiolated pea plants with red light and with red combined with far-red light produced morphologically similar plants having 4-fold differences in spectrophotometrically detectable phytochrome. Stem segments from the variously pretreated plants respond in the same way to different percentage conversions of phytochrome to P_FR. These results suggest that the P_FR./P_R ratio, rather than the concentration of P_FR, governs pea stem segment elongation. However, the ratio hypothesis does not explain contradictions between spectrophotometric and physiological assays previously obtained with this tissue, nor does it explain similar contradictions obtained in other systems. The only hypothesis consistent with the data to date is that of the existence of bulk and active phytochrome fractions, with the latter present in insufficient quantities to be spectrophotometrically detectable.     

Auxin inhibition of acid-and fusicoccin-induced elongation in lentil roots

Seedlings of the short-day plant, Chenopodium rubrum L. (Ecotype 60° 47 N) were irradiated with different intensities and qualities of light for 24 h preceding a single inductive dark period (12 h). Our data shows that a relatively low intensity incandescent light (35–100 ft. c.) is not effective as the photoperiod for flowering. The above effect is not due to a requirement for a relatively high level of photosynthesis. Our results suggest a definite promotory role of a blue High Energy Reaction (HER). We could not demonstrate the involvement of a far-red HER. We suggest that ineffectiveness of far-red may have been due to establishment of rather low Phytochrome, P _FR , levels, suboptimal for flowering. A certain critical level of P _FR (30–40%, that presumably established by blue light) seems to be necessary for photoreactions involved in flowering of C. rubrum. There are indications in our experiments of the operation of a red radiation mediated flower inhibitory photoreaction.Abbreviations SD short day plant - HER High Energy Reaction - P _FR far-red absorbing form of phytochrome - P _R red absorbing form of phytochrome - L.I.I. low intensity incandescent white light - H.I.I. high intensity incandescent white light - L.I.F. low intensity fluorescent white light - H.I.F. high intensity fluorescent white light - DCMU 3(2, 3, dichlorophenyl) 1, 1 dimethyl urea This paper constitutes a part of a Ph.D. thesis submitted to the University of Western Ontario, London, Ontario     

Action of Phytochrome During Prechilling of Amaranthus retroflexus L. Seeds

Dark germination of Amaranthus retroflexus L. seeds at 35° increased after several days of prechilling at 20° or lower. Irradiation with far-red light for short periods during the early hours of a prechilling period at 10° inhibited subsequent dark germination at 35°. The inhibition was completely reversible with red light. Far-red irradiation in the latter part of the prechilling period was less effective. Increased dark germination of A. retroflexus seeds following a prechilling period at 20° or less is attributed to action of preexistent P_FR, the far-red absorbing form of phytochrome, within the seeds. Inactivation of P_FR was found to proceed ca. 4 times more rapidly at 25° than at 20°. Failure of imbibition temperatures above 20° to increase dark germination of A. retroflexus seeds is attributed to the rapid thermal reversion of pre-existent P_FR. We suggest that the action of prechilling (layering) on many other seed kinds arises in a similar way.     

An action spectrum in the blue for inhibition of germination of lettuce seed

Summary Using a 2-h irradiation period at constant quantum irradiance, a complete action spectrum for inhibition of germination of lettuce seed has been obtained. Action maxima were near 470 and 720 nm, the latter being the most active wavelength. It was also shown, under conditions where light inhibition cannot occur, that phytochrome potentiation of germination is maximal at all wavelengths below 700 nm, including the highly active blue region. Evidence was presented for promotion of germination by a 2-h irradiation in the red which cannot be explained on the basis of conversion of phytochrome to the active form.Abbreviations Bl blue - FR far-red, P_FR far-red-absorbing form of phytochrome - R red Supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171 and the Graduate School Research Funds.     

Photomorphogenic and Chlorophyll Studies in the Bryophyte Marchantia polymorpha

Besides the standard rod (R) and far-red (FK) irradiations, a graded series of different R/FR ratios were tested as 10 min terminal exposures at the end of the daily 8-hour photoperiod of white fluorescent light. Water filtered incandescent light of 3780 lux during 10 min caused A rather weak hut reproducible effect. A superposition to the water layer of different filter combinations shifting the initial transmittance more towards the FR region, and thus gradually lowering the R/FB ratio, resulted in a parallel increase in orthotropic growth and a decrease in chlorophyll content. Our data show growth similarities with the results of other authors on light grown seedlings of higher plants. Rather high levels of the P_FR form of phytochrome seem to he required to maintain horizontal growth and optimal chlorophyll content in Marchantia thalli.     

Photophysiology of Kalanchoë Seed Germination

The effects of irradiations with different proportions of red/farred light and of gibberellic acid on the phytochrome-mediated seed germination of Kalanchoë blossfeldiana cv. Feuerblüte, were studied. The seed coat transmits much more red than far-red light, and therefore the energy ratio between 660 nm and 730 nm is given only for the transmitted light. Decreasing this ratio from 65 to 1.0 caused only a very slight inhibition. If this ratio is further lowered to 0.64, a 10 min terminal irradiation after a 3-h white light photoperiod is inhibitory, but a 12-h photoperiod or continuous irradiation is not. If the ratio is decreased to 0.44 or 0.31, a 12-h photoperiod is now also inhibitory, although continuous irradiation and 10 min terminal irradiation are still more inhibitory. These results are discussed in terms of phytochrome phototransformations. Although gibberellic acid is unable to cause any germination in complete darkness, it can result in a very high germination percentage, if combined with treatments which by themselves do not induce any germination such as continuous far-red, terminal far-red after short photoperiods, or very short photoperiods at 25°C. These results point to a strong synergism between gibberellic acid and the so-called stabilized form of phytochrome, P*_FR.     

Phototaxis in a dinoflagellate: Action spectra as evidence for a two-pigment system

Summary Action spectra were determined in the UV region of the spectrum for the first phase of the phototactic response (stop response) and for the phytochrome pigment associated with this response in the dinoflagellate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after FR irradiation they shift to 490 and 300–310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of P_R in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The P_FR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of P_R to P_FR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.Abbreviations FR far-red - R red - P_FR far-red-absorbing form of phytochrome - P_R red-absorbing form of phytochrome - UV ultraviolet     

Nature of light requirement for the flowering of Chenopodium rubrum L. (Ecotype 60° 47′ N)

Seedlings of C. rubrum were irradiated with different light qualities and intensities following a single inductive dark period. Our results show that relatively low intensity white light (35–100 ft. c.) does not support flower development while high intensity white light (650–800 ft. c.) permits 100% flowering. We have shown that the low intensity light inhibiton of flower development is not due to suboptimal photosynthesis. Relatively low intensities of light rich in far-red or blue wavebands sustains optimum flower development, whereas red light is totally ineffective in this respect. Considering that the intensity dependent High Energy Reaction (HER) has its action maxima in the blue and far-red we propose that HER may be positively involved in the flower development of C. rubrum. Our study further suggests that there may be some flower inhibitory component at play in relatively low intensity white light conditions and HER may be required to counteract this flower inhibitory effect.Abbreviations SD short day plant - HER High Energy Reaction - P_FR far-red absorbing form of phytochrome - P_R red absorbing form of phytochrome - L.I.I. low intensity incandescent white light - H.I.I. high intensity incandescent white light - L.I.F. low intensity fluorescent white light - H.I.F. high intensity fluorescent white light - GA₃ gibbrellic acid This paper constitutes a part of a Ph.D. thesis submitted to the University of Western Ontario, London, Ontario.     

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     

Inhibitory Action of Blue and Far-Red Light in the Flowering of Lemna paucicostata

In a new strain of short-day duckweed (Lemna paucicostata T-101), blue and far-red light-induced inhibition of flowering was investigated. Flowering of this strain failed to be induced under a short-day photoperiod of blue and far-red light, although it responded as a typical short-day plant in red and white light. When the short-day photoperiod of blue or far-red light was terminated by a 15 min red light pulse, flowering recovered completely. This inducing effect of red light was reversed by subsequent exposure to far-red light. Furthermore, it could be demonstrated that 30 min of blue light completely reversed the flowering inductive effect of 5 min red light and vice versa. Evidence is presented suggesting that the inhibitory action of blue and far red light may be due to the lowering of phytochrome P_fr levels below those required to start the dark reactions which lead to flowering. These results are discussed in relation to the time measurement system of photoperiodism.     

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     

Relationships between phytochrome state and photosensitive growth of Avena coleoptile segments

Using various photostationary state light sources to obtain reproducible phytochrome conversion of from 5 to 88% P_FR, assayed by 2 wavelength in vivo spectrophotometry, relationships between initial percent P_FR and elongation of apical Avena coleoptile segments over the succeeding 20 hours in darkness were studied. With material grown in total darkness, all P_FR levels promote elongation, and maximal promotion requires roughly 50% P_FR. The promotion caused by an initial 5 minute red (88% P_FR) treatment at hour 0 is partially reversible at hour 5 by sources forming less than 48% P_FR, but totally irreversible at hour 8, though less than 50% of the growth has been accomplished by this time. Direct photometric assays at hour 5 indicate a phytochrome state of roughly 45% P_FR, consistent with the reversal data. At hour 8, however, 11 to 22% of the phytochrome still assays as P_FR, an inconsistency suggesting simply that the elongation process has proceeded beyond photochemical control. Thus, in contrast with results previously reported for Pisum and Phaseolus, there is no contradiction between photometric and physiological assays of phytochrome state in Avena coleoptile segments.

Attempts to expand this study by using segments from seedlings pretreated with red light showed that such pretreatment as little as 1 to 2 hours before drastically reduces subsequent elongation and photoresponse on the medium employed. This decline in growth potential can be halted at any time before its completion by either excision of the segment or far-red treatment of the intact seedling.

     

The Role of Phytochrome in Anthocyanin Synthesis in Seedlings of Vigna radiata (L.) Wilczek

Previously, it has been demonstrated that the red light-inducedanthocyanin accumulation in mung bean seedlings is mediatedby phytochrome [Dumortier and Vendrig Z. Pflanzenphysiol. 87:313 (1978)]. In this paper the importance of phytochrome forthe accumulation of anthocyanins in seedlings of mung beanswas studied in non-irradiated seedlings and in seedlings irradiatedwith 5 min R. A short FR-irradiation given early after sowing reduced theamount of anthocyanins which were normally found in non-irradiatedseedlings. This indicates that P_FR may be important for at leastpart of the anthocyanin synthesis in the dark. As for the redlight-mediated anthocyanin accumulation, irradiation appearedto be most effective when given to seedlings at the age of 36–48hr. Although the seedlings were sensitive to red light irradiationbefore that time, they were not able to synthesize anthocyaninsuntil they had reached the age of 36 hr. Complete escape ofred/far-red reversibility occurred only when far-red was given12 hr after red, although partial escape could be observed witha shorter time-interval. Furthermore, the time-course of anthocyaninaccumulation after a two-fold R-irradiation was compared withthe effect of a single R-exposure. From the results could beconcluded that the pattern of anthocyanin accumulation is dependenton the time during which P_FR is present in the seedlings. Theseexperiments also indicate that P_FR not only plays a role inthe synthesis of anthocyanins but probably also in their degradation. The results of our study show that phytochrome is importantfor anthocyanin accumulation in non-irradiated mung bean seedlingsas well as in R-irradiated, and that it probably is also involvedin the degradation of the pigment. (Received January 18, 1982; Accepted April 30, 1982)     

Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary The substitution of red or blue light for the first six hours of prolonged irradiation with far-red light reduced anthocyanin formation by about 60%; red or far-red light similarly substituted for blue light had little effect. It is concluded that the effects of prolonged irradiation with blue and far-red depend, in part at least, on different photoreceptors.The effects of pre-treatment with red or blue light also occurred when only short exposures to light were given, and were reversed by immediate brief exposures to far-red. The depressing effect of a short pre-irradiation treatment was largely prevented if seedlings were kept at low temperature or in an atmosphere of nitrogen in the dark period before transfer to the prolonged far-red treatment. The effect of the pre-irradiation treatment is attributed to enzymatic destruction of phytochrome following conversion to the P _FR form, and it is suggested that anthocyanin synthesis in far-red light largely depends on phytochrome, possibly due to the maintenance of a low level of P _FR in the tissue by the absorption tail of P _R in the far-red.A pre-irradiation treatment with red also decreased the inhibitory effect of far-red on hypocotyl elongation but did not change the response to blue light.

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Zusammenfassung Die Anthocyanbildung war im langfristig gegebenen Dunkelrot bis zu etwa 60% reduziert, wenn die ersten 6 Std durch hellrote oder blaue Bestrahlung ersetzt wurden; Hellrot oder Dunkelrot in gleicher Weise im Dauerblaulicht substituiert waren praktisch wirkungslos. Daraus wird geschlossen, daß der Effekt einer Dauerbestrahlung mit Blau und Dunkelrot zum Teil jedenfalls, auf verschiedene Photorezeptoren zurückzuführen ist.Der Effekt einer Vorbehandlung mit hellrotem oder blauem Licht trat auch dann auf, wenn nur kurzfristige Bestrahlungen gegeben wurden und konnte durch unmittelbar nachfolgende kurze Dunkelrot-Belichtung wieder aufgehoben werden. Die Hemmung durch kurzfristige Vorbestrahlung konnte weitgehend verhindert werden, wenn die Keimlinge während der Dunkelperiode, vor der Übertragung in Dauerdunkelrot, bei tiefer Temperatur oder unter Stickstoff gehalten wurden. Der Vorbelichtungseffekt wird auf die enzymatische Destruktion von Phytochrom, nach der Umwandlung in die P _FR -Form, zurückgeführt und es wird vermutet, daß die Anthocyansynthese im Dauerdunkelrot weitgehend phytochromabhängig ist, wahrscheinlich durch die Aufrechterhaltung eines niedrigen P _FR Niveaus im Gewebe infolge der schwachen Absorption von P _R im Dunkelrot.Eine Vorbelichtung mit Hellrot verringerte ebenfalls die hemmende Wirkung von Dunkelrot auf das Hypokotylwachstum, war jedoch ohne Einfluß im Blaulicht.

     

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.     

Immediate phytochrome action in lettuce seeds and its interaction with gibberellins and other germination promoters

Summary An immediate action of phytochrome (P_FR) was found by a technique of adding subthreshold concentrations of gibberellic acid (GA₃) after irradiation. It was shown that phytochrome (P_FR) is active within 5 mins after its formation. The interaction between P_FR, GA₃ and six other gibberellins (GA₁, GA₄, GA₅, GA₇, GA₉ and GA₁₃) was synergistic. This result is interpreted to mean that P_FR does not produce any of these gibberellins, at least during the first 30 mins. of its action. This conclusion was confirmed by interaction experiments in darkness using GA₃ combined with the other gibberellins. A similar synergism was found between P_FR and kinetin, thiourea and chloramphenicol.     

Some Properties of Phytochrome Isolated From Dark-grown Oat Seedlings (Avena sativa L.)

Phytochrome was partially purified from etiolated seedlings of Avena sativa L. Several properties of the red-absorbing (P_R) and far-red absorbing (P_FR) forms of the pigment were compared. The 2 forms could not be shown to differ with respect to their sedimentation velocity in sucrose density gradients, elution volume from Sephadex G-200 columns, binding properties on calcium phosphate, or electrophoretic mobility. P_FR, however, was more labile than P_R during precipitation with 50% ammonium sulfate. Sephadex G-200 elution diagrams obtained with fresh phytochrome preparations revealed 2 components of different molecular weights, 1 roughly 180,000, and 1 roughly 80,000. Native phytochrome had an absorption spectrum in vivo showing an absorption maximum for P_R of 667 nm. Both the large and small forms of phytochrome mentioned above can be maintained with an absorption maximum for P_R of 667 nm. However, allowing them to remain for several hours as P_FR, even at 4°, shifted this peak to 660 nm. The protein conformational change during phytochrome transformation may be quite small, though the various comparative techniques used do not strictly rule out a fairly large one. The need for maintaining the pigment as P_R during all steps of purification, but particularly during ammonium sulfate precipitation is underscored.