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Regeneration dark grown of protochlorophyll seedlings illumination with in following red and far red light C.J.P. Spruit Laboratorium and C.W. Raven Plantenfysiologisch Onderzoek, Landbouwhogeschool, Wageningen.* voor SUMMARY The rates of regeneration of protochlorophyll (Pchl) pea and maize far red. In neither rates of pigment final level of studied, following were material reached sequent to a effect of continuous latent in decreases than after far red. ded that the found we evidence red formative leaves, less for an rapidly during the red effect far red - was not due to a of red - far red control during of the of Pchl red light. the rate earlier part of a of terminal It is concluin sub- greeningprocess regeneration by initial upon the only plant bean, followed regeneration after of the the red but that in this stages the kinetics becoming manifest process, last or control observed of seedlings grown alone, pigment photobleaching in pre-illumination upon light, is of dark prolonged darkness, indicating There is considerable a in leaves dose of either red saturating In older maize regeneration. Pchl, pigment production red have a as but such, subsequent prolonged light period. 1. INTRODUCTION Chlorophyll In and is be phyllide the as dark grown under supposed and seedlings remains in the biosynthesis to be to far required below and subsequent the has plant evidently of this compound, transfer protochlorophyllide. rate the of Chi a formation (Price & Klein 1964). Usually, * 283rd Communication Acta Bot. Neerl. a the 19(2), April a other steps no a, relatively a repressing since 1961; low pigment an in reached proto- early step other intermediates have been A brief illuminationof dark treatment Mitrakos Plant to protochloro- level of effect upon to grown chlorophyllide formation of additional by the strongly dependent initial upon of the dark grown material. 1961; Henshall & Goodwin the dark grown plants for 1960). appear ultimately protochlorophyllide light Laboratory 1970 All second illumination is experiments of a. level of Chi of plants. the first Virgin 1961; in these delta-amino- reports in the literature indicate that during light quality, given during via Bogorad (e.g. for the transformation of the darkness is followed to Several formed The concentration of this Build-up accumulate in normal be reared in the dark accumulate material results in transformation of the a to precursors chlorophyllide to continuous illumination. reported supposed tetrapyrrole temporary end product. plants chlorophyllide in the of protochlorophyllide light independent is plants series a Angiosperms, light precursor in (Chi a) a acid levulinic Physiological were pretreated either Research 165 C. with red alone, of Chi formation in continuous a far red during of the 1958; depends (Virgin of this phase by 1961). These observations of Pchl. During etiolated pea by preliminary far red. in small vitro Pchl at size of the pigment have included in we considerably are this In the article, forms present phyllide these ditions of Therefore, that we have represent phytylated MATERIALS AND 2.1. 85% was and be a this steady pretreatment. regeneration illumination of rates of Pchl either red material these in two with contrast or the light the red some rates treat- findings, Since pea, because of the darkness, well as Pchl. as the rather suitable particularly a at made Maize terms material, which seedlings, was for the chosen a as intend and a pigment separate paper. It a an fraction of 1957), pigment may in absorption a’’ denotes the fate of under the forms, non- protochloro- discuss to and be relevant to pigment(s) having We nm. found form of the phytylated appear Likewise, “chlorophyll non-phytylated in not protochlorophyll acetone. 665 between this did by light (Shibata be stated con- here, protochlorophyll our and that this fraction appears (Sironval et al. 1965). METHODS of Pisum sativum Zea mays rel. cv. the cv. “Caldera” hum. Seeds added in experiments 166 the Plant material Seedlings and 80% invariably to 2. in present experiments, be non-transformable not rich since promiscuously to a will the mixture, absorbing our of leaves of dark grown bean pigments, phytylated subsequent nm state distant taxonomic group. distinction nm is in study. grown in especially about 660 at evidence and the results of extensive weight, the peak different for rate of which length reactions after form to seems more seedlings, and more about 628 various however, no a in the duration became interested in the we al. el elimination of the saturating irradiation with spectroscopic this study of the context. at pigment the a dark significantly unit per our have been used maximum of larger from representative phytylated sight leaves a for- a (Withrow is involved in phytochrome 1967), not undertook content that rate cases, of Chi rate the the a the estimations indicated that were first we for independent be phytochrome Both direct regeneration quoted above, low of seedlings (Spruit Since ments. might suggest study the period, mainly phase, spectrum lag phase, to appears in this materialafter regeneration followed a induction of the W. RAVEN in continuous illumination: the a an AND C. number of a hours after the first short illumination has completion formation dark a This manifests itself action The After measured. In was some shows preceding a red. SPRUIT slow down the initial to above). pigment At the pigment of of Pchl also see upon pretreatment. induction rate reported was far by light in the formation of Chi lag phase production followed illumination, given an Virgin 1956; of after red given mation with red or J. P. were dark consisted “Krombek”, were sown during of the in the Phaseolus vulgaris reared in total darkness pasteurized soil. growth period. “plumules” of the As far at as cv. “Widusa” 20 °C and about necessary, water The leaf material used in the pea (third Acta Hot. and Neerl. fourth unex- 19(2), April 1970 REGENERATION PROTOCHLOROPHYLL internodes with panded the OF the bean and vesting the done in absolute was 2.2. Irradiation The phototransformation the of light filters and 2.9 2.3. Leitz a 4 10 x 10 x of Pchl erg/cm erg/cm 2 was able. We used a fluorescent tube moid” nr. 62 moid” nr. 46. at sec slide descent lamp, und 735 529 2.4. 2.2 nm, Alternatively, the by intensity 4 10 X of interference means The erg/cm were layers stat 25 °C for the the at 2 at sec samples 650 nm for 5 minutes. given gram in placed petri leaves appropriate treatment, of calcium carbonate. The filter with 80% milliliters of the minutes with suction. 12 ml up The residue acetone-water. same at solvent. 60.000 a few 5 nm cm nm were as well as the of the at path length of “Cine- 25 W incan- a blue “Plexiglass” 46. Precautions nr. samples directly. light-tight 800 scatter. and and the 2°C, flasks nm cells. the on If in a to was lined were tins in a thermo- extracted filter filter was were extracts kept the the rarely in again and filtration extracted with washed then in the % at with few a optical the to more as 600 region model density readings wavelengths amounted to For storage 2°C. throughout spectrophotometer other a centrifuged acetone. dark a over clear supernatant transferred Beckman at by were 0.01 by grinding sand, a on nearest washed removed was possible as the to little a measured necessary, This correction 311) volume with 80 were readings quickly as were liquid Vessels extracts subtracted from the a trace mm reach the model The combined g and hours, optical density 700 using to in pure acetone, ml volumetric flask and made up The put samples amount periods of dishes the lids of which Corp. small 25 not weighed were Scale (Ohaus together for 30 did were mortar ml 3 of blue “Cine- orange-yellow required periods. balance gram. After the glass of through monophosphor layer of “Cinemoid” sources small about 7 a used, consisting filtered layer found unavoid- green in layer a was regeneration were of the samples “Centogram” a estimations Pigment One and was of moist filter paper. These with 4 2.5. one from these light Protochlorophyll at Corp.) was light of 17), wrapped safelight a plus 0248 nr. little a safelight, consisting of which light Haas) samples, TL 40, colour Strand Electric The irradiated samples for projector All radiations nm. of the green (Philips taken that the were to har- bands isolated from spectral Filtraflex B 40). 2 handling weak (The (Rohm a of pair of maize. All Safelights During subsequent a leaf primary coleoptiles effected with 500 W at sec the material), whole darkness. “Prado” 4 1.4 leaf the or (Balzers, Liechtenstein, type about was attached leaves primary a DU, at 800 correction than 0.01 O.D. units. Acta Bot. Neerl. 19(2), April 1970 167 C. J. 2.6. The rather This is sensitive a small to Fig. wavelength errors, and the by the Boardman (1962). during checked was each calculated values are spectrometer calibrations dependent. 1 gives the concentrations of total Pchl in bean leaves period following Pchl regeneration confirming seedlings, a red saturating proved or red - was pigment than following 5 rate of material, of Akoyunoglou & Siegelman (1968). In young dependent appears to periods up jxg; somewhat different the we of original values scatter to the level before approach 24 hours, no rate significant or the effect final level or 5 min. picture (fig. 2). Regeneration Pchl level. Because of the small in bean leaves red plus 5 min. of of Pchl rate varying far red. of phototransfortned protochlorophyll. The days, amounts ± 23 ug; of 21 pigment days, ± per 23 gram fresh regeneration during age, Results a expressed 100% level weight: 8 day never amounts within the Nevertheless, considerably. difference in the no protochlorophyll absolute 14 the observed min. red of the amounts following a 50% of formed, accuracy, 12 The of the During regeneration Regeneration ± function of the found. more limits of a upon the age strongly plateau ultimately reached Pea leaves present reached as far red irradiation. terminal far red irradiation upon either the initial of Pchl Fig. I. a the observations the illumination. 168 regeneration Protochlorophyll dark of RAVEN RESULTS 3.1. of since precaution, necessary somewhat temperature proved coefficients used absorption calibration of the spectrophotometer wavelength AND C. W. concentrations pigment the molar adopted measurement. 3. of Calculation We have P. SPRUIT dark as period cent per represents old the seedlings, |xg. Acta Bot. Neerl. 19(2), April 1970 OF REGENERATION Fig. 2. PROTOCHLOROPHYLL Regeneration to 1. The fig. of protochlorophyll in 100% level represents leaves a of 7 day old pea pigment regeneration See also seedlings. of ± 3.5 (xg per legend gram fresh we'ght. Fig. 3. Regeneration various ages. along the The 9 or ± 3.5 axis. the See |xg; 14 red followed days, by ± far In maize of various ages the increase in rapid Acta Bot. Neerl. legend 19(2), April maize curves to fig. of the 8 (xg; 17 days, ± at 8 :xg; a Pchl, no In 21 ± days, seedlings was significant dark in ages leaves are of shifted gram fresh weight: 7 (xg. between 5 and 14 low level. value in 9 original and 1. red in pea also. Upon longer 1970 coleoptiles for the successive following pigment regenerations per {fig. 3), regeneration 80% red-far red could be found. old day the uniformly stopped final level of about of 9 reading, further represents the days old, regeneration period in protochlorophyll vertical 100% level days, after red of To facilitate fairly rapid, day old leading differences between red incubation, to a coleoptiles. During a and difference between 169 C. Fig 4. Absorption spectra About a. b. 1 the light two of about cally g bean 17 3.2. In amounts red following in Chlorophyll confirmed the grown in complete a in of the the days, dark 1 cell 1 in 80% final level, about red most RAVEN acetone, under the optimum final level of be not in leaves way, is statisticonditions. Pchl reached demonstrated, whereas in of the effect decreased. grown leaves contain a (Spruit substance that leaves 1966) spectroscopically 4. The concentration amounts but W. C. marked reached in this 25% decreases magnitude significant Pchl present, AND cm. apparent which is to SPRUIT cm. earlier observation darkness, and bean pea this effect could coleoptiles, We have Chi becomes and 17 grown P. cm. cell The difference in older than leaves, cell 5 Absorption treatments that far in darkness. of dark seedlings. Absorption pea Absorption leaves. days. significant, It shows of extracts whole g leaves. g pea 4.2 c. 100 J. there are to of pea, similar roughly 25 to % considerable variations between individual batches. In experiments, most this sowing. During diffuse daylight. tained the To under sown pigment therefore does as whether green as in water treatment control batches safelight. same The proportion of the seeds responsible for a few during sowing, they this seedlings, our Exposure appear soaked well were for to the imbibed in absolute likewise con- in the material treated in light during to for the before exposed responsible was leaf material as hours were the imbibition “chlorophyll” period found in the dark seedlings. Trace amounts controls of 170 not were in about the the usual way ,fig. 4. grown check found in the “chlorophyll” darkness and peas treatment our of a chlorophyll-like bean and maize substance could be observed in the dark experiments, but the quantities Acta Bot. Neerl. were very small 19(2), April 1970 REGENERATION and OF PROTOCHLOROPHYLL of doubtfull this respect. significance. It appears plant originates in the green the early stages of 3.3. Yield of Obviously, have We the estimated both the Fig. 5. loss Yield at 0° due to some was of in Pchl observed measure of a this light difference of loss spectrum a min. red. It must bleaching of 25°. a embryo during be likely in green. At 0° primary Smith in optical stated here that the form of Chi a at at 2 0°. the pigments pigment absorbing loss at vivo. caused pigment old bean day leaves 6 an (1961) also attempted gives were by for the yields an to example preirradiated one min. red. at The additional dose of changes can be very forms may be involved. have observed is due about 685 is lost relatively higher have Fig. by pigment we of 8 We a. absorption or a lower than Goedheer The leaves density, is a. Pchl separate sample, 25°. Similar min. followed that a of of irradiation. (1948). in mole of Chi temperature effect is leaves duration of the by spectrophotometry for this likely, formed Chi for bean leaves one formed and a fraction of the a with Most function initially and it appears that several At any rate, it is in position leaves of this quantities two the illumination temperature with green light complex 4. at so reported by were photobleaching pigment as during of Pchl spectrum gives the changes 9 less light, observed than regeneration this the to is derived from figure protochlorophyllphotoconversion photoconversion in the give of Chi scatter. It is evident that green and red should by light quantity in this points illumination with red pigment transported the ratio of these irradiation, 5. As each of the show different a pigment photoconversion mole of Pchl converted always points during and is cotyledons protochlorophyll one occupies pea that the possible, development. converted. After red one,fig. Evidently, least at to photo- nm. DISCUSSION Our results upon Pchl have failed observations with oat Acta Bot. to regeneration (Spruit show during 1967). significant the first effects of the hours of A similar result was phytochrome darkness, obtained confirming system earlier by Jacques (1968) seedlings. Neerl. 19(2), April 1970 171 C. J. P. SPRUIT Fig. Difference 6. luminated sent the In young first hour of bean, This led and the initial It is final by be an nature far red illumination, fig. for Chi of the have a pea and in due If far red 3. a nm. All preilrepretreat- period longer that is the also possibly few hours. a dark periods. depressed by conclude must in the during constant is photoperiod that not a the itself a we want fit in with to those, demonstrating subsequent a continuous illu- falls somewhat outside the scope make a few remarks that may aid in problem. reported that the light by given immediately a after red 1968). According to maize leaves. red. With We to can of the shortening lag pre-exposure (Withrow Virgin al. (1961), findings 1956; in wheat, in the can be Mitra- in this far red is antagonism red-far red bean, far red reversal of the our red, Henshall & Goodwin 1963; confirm the el period to red matter effect was will appear course. Pchl, 172 We brief during problem however. A detailed report of we assume rate rate was spectrum induction an darkness, findings formation in continuous weakly antagonistic doubtful, of following clear how these of Akoyunoglou 1964; material min. at 653 9 essentially 1961; Price & Klein 1961; Sisler & Klein only the formation is regeneration accumulation of Chi by evidence for or no This aspect of the Several authors kos for The difference induction in Pchl regeneration lasting of the present article. However, eliminated The RAVEN red-far red mechanism. control the illumination level after 24 hours pigment the mination period. defining nm. red-far red effect could be observed after immediately red-far red bean. grown 653 the irradiation. In older leaves of maize and of Pchl rate not a terminal controlled of dark min. at additional of Pchl rate seems to only, to a an 1 plus there is little material, there preceding during nm C. W. 0°. following In maize leaves primary min. at 529 changes ments at regeneration of spectrum for 2 AND of Chi that the a rate which condition of Chi a of Pchl formation as such is accumulation should equal the initial clearly is accumulation during not not rate influenced of dark by light, regeneration satisfied. In many experiments the initial illuminationis much lower than the initial Acta Bot. Neerl. 19(2), April 1970 REGENERATION of rate after OF PROTOCHLOROPHYLL Pchl pigment to converted Chi to dark grown Pchl, light and, troyed by light, the of the capacity with the first stages of accumulation is mechanism may the account pigment red effect. lag short and irradiation, provision may, in the that is, are they manifest therefore, overall rate Pchl saturate photoproducts the Pchl des- are of rate Chi a concomitant increases, a net of Chi rate a of Pchl formation. This rate observed accumulation, of the influence of light We have started work are points during during only suppose the re- biosynthetic some for a by way red the prolonged subsequent that these effects represent either from pigments and of Pchl rate the of attack in prepared Chi pigment forming intensity of development photodestruction of or a, these to wavelength a formative the during system experimental approach An influence the to possible accumulation of of the capacity prolonged irradiation. study original pre-illumination photodestruction We phase the regulating the to of Pchl formation. In continuous rate mechanism, protecting photoreversible the a and become irradiationperiod. ring initial intensity, for the kinetics of Chi further a The pre-illumination a surpassing a formed follows the a sufficient biosynthetic system forming reached, apparatus during -far upon of Chi production of the of Chi newly already present, is rapidly light intensity less than the of rate initial continuous illumination. by Pchl chloroplast development. Ultimately, the failure of generation, the light kinetics photobleaching biosynthesis time, part same accumulation a severalfold the attribute the to initial further a Chi and afterwards. lag phase Given at depending is, therefore, accumulation inclined an the rule surpasses a material, on, least at At the photoconversion. a From then a. of production are as stimulation of Pchl light with whereas dark, combination of a Upon irradiation of of We regeneration. accumulation the of illumination hours of Pchl rate in regeneration several subsequent may be the problems upon Chi du- effect, a accumulation. this line. along REFERENCES Akoyunoglou, in Hanover p. , — G. A. dark-grown & H. W. Plant Siegelman 43: N. K. tive L. effect (1960): J. C. I. properties. and far-red Abstr., ages. light Fifth on chlorophyll biosynthesis Intern. (1968): Protochlorophyllide resynthesis Studies on a determination. Congr. in PhotobioL, on dark-grown The (1961): biosynthesis Effect Fluorescence of protochlorophyll-protein complex. I. Biochim. biochemistry ofphotoreactive Goedheer, of red various at bean leaves. 66-68. (1962): molecular-weight Bogorad, leaves 86. Physiol. Boardman, The (1968); bean of Acta 62; protochlorophyll. systems. changes and Biophys. Acad. in Press, chlorophyll of absorption In: M. New B. Allen (ed.). Compara- York-London, concentration greening Purification and 63-79. bean on leaves. 227-256. photosynthetic Biochim. Biophys. Acta 51: 494-504. Henshall, and Jacques, tion, 6: J. D. & T. W. Goodwin chlorophyll R, la (1968): formation Action croissance et le in pea de la (1964): The seedlings. lumière par effect of red and far-red Photochem. PhotobioL l’intermédiaire du light on carotenoid 3: 243-247. phytochrome développement de Chenopodium polyspermum L. sur la germina- Physiol. Végét. 137-164. Acta Bot. Neerl. 19(2), April 1970 173 C. Mitrakos, K. (1961): metabolism. Price, The Physiol. participation Plant. L. & W. H. Klein 14: of the red far-red J. P. SPRUIT reaction AND C. W. RAVEN in system chlorophyll- 497-503. (1961): Red, far-red response & chlorophyll synthesis. Plant Physiol. 36: 733-735. Shibata, (1957): K. Biochem. (Japan) Sironval, C., functions M. R. of the studies Spectroscopic 44: Michel-WolWertz 673- chlorophyll on formation in intact leaves. J. 147-173. and 684- mp, & A. forms Madsen in vivo of On the nature (1965): Biochim. chlorophyll. and possible Biophys. Acta 94: 344-354. Sisler, of E. C. & W. H. Klein chlorophyll synthesis J. Smith, H. C. (1948): (1963); in dark The effect of bean grown Protochlorophyll, age and various chemicals seedlings. Physiol. of precursor Plant. chlorophyll. on 16: the lag phase 315-322. Arch. Biochem. 19: 449-454. C. Spruit, In: J. P. Currents (1966): Spectral schoten, Zeist, 1965, — (1967): Virgin, Meded. (1958); H. I. varying light — (1961): in Action R. synthesis chromatic 174 B., in Studies J. grown and reversal on the West.-Eur. in leaves for the leaves Plant bean of 347-362. elimination of wheat. leaves Physiol. and 67: Plant. 11: 31: of a suppi.: antagonism in plants. Woud- Photosynthesis, on stem (14): sections the lag phase Plant. Elimination pretreatment of etiolated pea 1-6. protochlorophyll Physiol. (1956): by red-far red Conf. 67-74. formation B. Wolff & L. Price dark-grown energy. to the related Sec. Donker, A., Rotterdam, Physiol. spectrum dark Proc. Landbouwhogeschool Wapeningen treatments. previously Withrow, Publ. Phytochrome decay seedlings. observations photosynthesis, in and in chlorophyll chlorophyll a under formation 14: 439-452. of the with lag phase low of chlorophyll irradiances of mono- XIII. Acta 801. Neerl. 19(2), April 1970