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.     

Rapid recovery of photosynthetic cell suspension cultures following heterotrophic bleaching

Summary Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes.     

Characteristic symptoms of photosynthesis inhibition by herbicides are expressed in photomixotrophic tissue cultures of Nicotiana

A photomixotrophic tissue culture system for Nicotiana plumbaginifolia and N. tabacum has been developed in which a primary symptom (bleching) of the inhibition of photosynthetic electron transport by herbicides can be observed. Photomixotrophic cultures were initiated and maintained in the light on medium containing 0.2–0.3% sucrose or glucose (low-sugar medium) as sole source of respirable carbohydrate. The usual medium for growing heterotrophic cultures contains 2–3% sucrose or glucose (high-sugar medium). Callus grown on low-sugar medium achieved a fresh weight three to four times greater in the light than in the dark and reached about half that of callus grown on high-sugar medium. Carbon-dioxide fixation rates were an order of magnitude higher in cultures grown on low-sugar medium in the light than in those grown on high-sugar medium or in any of the dark-grown cultures. The lightdependent growth and CO₂-fixation rates of cultures grown on low-sugar medium indicated that a major proportion of the weight increase resulted from photosynthesis. Under these photomixotrophic conditions it was found that a number of photosystem-II herbicides, at concentrations which inhibit photosynthetic electron transport, also inhibited the light-dependent component of callus growth, and caused bleaching. These effects could not be demonstrated on high-sugar medium.Abbreviations PSII photosystem II For common names of the herbicides the reader is referred to Weed Res. 19, 401–406 (1979)     

A comparison of light-dependent RNA metabolism in wild-type Euglena with that of mutants impaired for chloroplast development

Summary The possibility that ³²PO ₄ ^3- (³²P_i) labeling of both chloroplast and non-chloroplast RNAs during light-induced chloroplast development in Euglena is due, in part, to the break-down of existing RNAs and their resynthesis into labeled RNAs has been examined by comparing the RNA content of dark-grown, non-dividing cells after completion of light-induced chloroplast development with that of identical cells maintained in darkness for the same period of time. The involvement of the photo-conversion of protochlorophyll to chlorophyll and other photoreceptor systems in the labeling of RNA during chloroplast development has been considered by comparing the labeling pattern obtained with wild-type cells with the patterns obtained with mutants of Euglena which either lack detectable amounts of protochlorophyll and chlorophyll or form only rudimentary chloroplasts upon light induction.No significant difference in RNA content between dark-grown, non-dividing cells containing fully developed chloroplasts and the same cells maintained in darkness for the development period can be detected. This observation is interpreted to mean that in non-dividing cells precursors for chloroplast-associated RNAs are derived from pools and pre-existing RNAs, including non-chloroplast RNAs, and that the matebolic entrapment of ³²P_i involves a light-dependent turnover and DNA-directed RNA synthesis in wild-type cells.The RNA profiles on sucrose gradients of mutants of Euglena show no remarkable deviation from the profile established for wild-type cells. The labeling patterns obtained after 24 hours of incubation in light and in darkness differ from that obtained for wild-type cells in that all mutants show less of a light-minus-dark difference than wild-type and that mutants lacking plastid-associated DNA and detectable amounts of chlorophyll incorporate considerably more ³²P_i into RNA in darkness than wild-type. One such mutant shows no significant difference in its light-dark labeling pattern.These observations indicate that cells possessing normal proplastids capable of forming functional chloroplasts regulate metabolism of RNA in darkness in a different manner than with either rudimentary chloroplasts or containing no detectable plastids structures. The possible involvement of more than one photoreceptor system in metabolic control is discussed.Supported by a grant from the National Institutes of Health, GM 14595     

Stability of phytochrome concentration in dicotyledonous tissues under continuous far-red light

Summary The phytochrome concentration in dark-grown seedlings of Pisum sativum, Phaseolus aureus and Sinapis alba remained constant under continuous far-red illumination for periods of up to 6 hours. Similar treatment of Zea mays seedlings reduced the phytochrome concentration by more than 60 percent. The results in the dicotyledonous seedlings may be due to the reversion of P_fr to P_r at a rate sufficient to prevent P_fr destruction; no evidence for reversion has been detected in Zea. Typical photomorphogenic responses were observed in the dicotyledonous seedlings in the absence of P_fr destruction.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

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])     

Action of light on accumulation of carotenoids and chlorophylls in the milo shoot (Sorghum vulgare Pers.)

We have performed a comprehensive study on the mechanism of regulation of carotenogenesis by light in the shoot of Sorghum vulgare. Our work shows that carotenoid accumulation is simultaneously controlled by phytochrome (P_fr) and by the availability of chlorophyll. Throughout plastidogenesis light dependent chlorophyll and carotenoid accumulation are interdependent processes: Accumulation of chlorophyll in natural light requires the presence of carotenoids; likewise, accumulation of considerable amount of carotenoids depends on the availability of chlorophyll. However, in both cases the efficiency of the biosynthetic pathway, the potential biosynthetic rates (capacities) are determined by phytochrome. A push and pull model of carotenogenesis advanced previously (Frosch and Mohr 1980, Planta 148, 279) to explain carotenogenesis in the mustard (Sinapis alba) seedling also applies to the monocotyledonous milo (Sorghum vulgare) seedling. Therefore, we suggest that the model applies to carotenogenesis in higher plants in general.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR High irradiance response (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P red absorbing physiologically inactive form of phytochrome - P_tot total phytochrome - i.e. [P_r]+[P_fr] =[P_fr]+[P_tot], wavelength dependent photoequilibrium of the phytochrome system - RL red light - FR far-red light     

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

For dark-grown seedlings of Pharbitis nil capacity to flower in response to a single inductive dark period was established by 24 h white, far-red (FR) or ruby-red (BCJ) light and by a skeleton photoperiod of 10 min red (R)-24 h dark-10 min R. FR alone was ineffective without a brief terminal (R) irradiation, confirming that the form of phytochrome immediately prior to darkness is a crucial factor for flowering in Pharbitis. The magnitude of the flowering response was significantly greater after 24 h FR or white light (WL) (at 18° C and 27° C) than after two brief skeleton R irradiations, but the increased flowering response was not attributable to photosynthetic CO₂ uptake because this could not be detected in seedlings exposed to 24 h WL at 18° C. Photophosphorylation could have contributed to the increased flowering response as photosystem I fluorescence was detectable in plants exposed to FR, BCJ, or WL, but there were large differences between flowering response and photosystem I capacity as indicated by fluorescence. We conclude that phytochrome plays a major role in photoresponses regulating flowering. There was no simple correlation between developmental changes, such as cotyledon expansion and chlorophyll formation during the 24-h irradiation period, and the capacity to flower in response to a following inductive dark period. Changes in plastid ultrastructure were considerable in light from fluorescent lamps and there was complete breakdown of the prolamellar body with or without lamellar stacking at 27 or 18° C, respectively, but plastid reorganization was minimal in FR-irradiated seedlings.Abbreviations BCJ irradiation from photographic ruby-red lamps - FR far-red light - P_fr far-red-absorbing from of phytochrome - P total phytochrome content - R red light - WL white light from fluorescent lamps     

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.     

Evidence that a mustard seedling responds to the amount of Pfr and not to the Pfr/Ptotratio

Abstract Phytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) was investigated. Light pre-treated and dark-grown seedlings differing in responsiveness and level of phytochrome (P_tot) were compared. The data obtained support the traditional view that a seedling measures the amount of P_fr. The alternative view that a plant measures the P_fr/P_tot ratio does not seem to be compatible with the data obtained with the mustard seedling.     

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.     

Control of chlorophyll synthesis by phytochrome

Summary The rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (P_fr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that P_fr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by P_fr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55–58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of P_fr.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Chl(ide) sum of Chl and Chlide - PChl protochlorophyll - PChlide protochlorophyllide - PChl(ide) sum of PChl and PChlide - P_fr far-red absorbing form of the phytochrome system     

The role of phytochrome in photoperiodic time measurement and its relation to rhythmic timekeeping in the control of flowering in Chenopodium rubrum

Summary To follow changes in the status of phytochrome in green tissue and to relate these changes to the photoperiodic control of flowering, we have used a null response technique involving 1.5-min irradiations with mixtures of different ratios of R and FR radiation.Following a main photoperiod of light from fluorescent lamps that was terminated with 5 min of R light, the proportion of P_fr in Chenopodium rubrum cotyledons was high and did not change until the 3rd hour in darkness; at this time, P_fr disappeared rapidly. When the dark period began with a 5-min irradiation with BCJ or FR light to set the proportion of P_fr low P_fr gradually reappeared during the first 3 h of darkness and then disappeared again.The timing of disappearance of P_fr is consistent with the involvement of phytochrome in photoperiodic time measurement. Reappearance of P_fr after an initial FR irradiation explains why FR irradiations sometimes fail to influence photoperiodic time measurement or only slightly hasten time measurement. A R light interruption to convert P_r to P_fr delayed, the timer by 3 h but only for interruptions after and not before the time of P_fr disappearance. Such 5-min R-light interruptions did not influence the operation of the rhythmic timekeeping mechanism. Continuous or intermittent-5 min every 1.5 h-irradiations of up to 6 h in duration were required to rephase the rhythm controlling flowering. A skeleton photoperiod of 6 h that was began and terminated by 5 or 15 min of light failed to rephase the rhythm.The shape of the curves for the rhythmic response of C. rubrum to the length of the dark period are sometimes suggestive of clocks operating on the principle of a tension-relaxation mechanism. Such a model allows for separate timing action of a rhythm and of P_fr disappearance over the early hours of darkness. Separate timing action does not, however, preclude an interaction between the rhythm and phytochrome in controlling flowering.Abbreviations FR far-red - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - R red - BCJ photographic ruby-red irradiation A grant in aid of research from the National Research Council of Canada to B. G. Cumming is gratefully acknowledged.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Phytochrome decay in seedlings under continuous incandescent light

Summary Under continuous high intensity incandescent light the decay of phytochrome in Amaranthus seedlings deviates from the predicted first order rate characteristic of the P _fr/P _total ratio maintained. This deviation takes the form of a slower decay than would be predicted and is only observed at high intensities. Experiments are presented to test the hypothesis that this reduced rate of decay is the result of a high level of phytochrome intermediates maintained under high intensity incandescent light. Accumulation of intermediates under these conditions has been demonstrated using a quasi-continuous measuring spectrophotometer. They are weakly absorbing and their concentration increases with light intensity. Although they form P _fr in darkness, it is proposed that they do not decay. The model predicts that in a sample cuvette, where a light intensity gradient exists, there is more probability of a phytochrome molecule being presnet as P _fr at the back of the cuvette: the region of lowest light intensity. Under conditions which favour phytochrome decay, a preferential loss of phytochrome should result at the back of the cuvette and an increasingly higher proportion of the remaining phytochrome will consequently be measured as intermediate as the experiment progresses. The results confirm the hypothesis and in addition, after 60 min incandescent light, demonstrate an accumulation of intermediates which form P _fr with a longer half-life that at the begining of the experiment. Pisum epicotyl hooks show no such intermediate accumulation or preferential decay at the back of the cuvette, which is in agreement with the observed first order phytochrome decay under high intensity incandescent light. A scheme is presented explaining the results on the basis of the decay process.Abbreviations FR far-red light - R red light - P phytochrome - P _fr far-red-absorbing form of P - P _r red-absorbing form of P 321st communication of this Laboratory.     

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     

Far-red induced changes of the protochlorophyllide components in wheat leaves

Biosynthesis of chlorophyll is partly controlled by the phytochrome system. In order to study the effects of an activated phytochrome system on the protochlorophyllide (PChlide) biosynthesis without accompanying phototransformation to chlorophyll, wheat seedlings (Triticum aestivum L. cv. Starke II Weibull) were irradiated with long wavelength far-red light of low intensity. Absorption spectra were measured in vivo after different times in the far-red light or in darkness. The relationship between the different PChlide forms, the absorbance ratio _{650nm636 nm} changed with age in darkness, and the change was more pronounced when the leaves were grown in far-red light. Absorption spectra of dark-grown leaves always showed a maximum in the red region at 650 nm. For leaves grown in far-red light the absorption at 636 nm was high, with a maximum at the 5 day stage where it exceeded the absorption at 650 nm. At the same time there was a maximum in the total amount of PChlide accumulated in the leaves, about 30% more than in leaves grown in darkness. But the amount of the directly phototransformable PChlide, mainly PChlide_650–657, was not increased. The amount of PChlide_628–632, or more probably the amount of (PChlide_628–632, + PChlide _636–657) was thus higher in young wheat leaves grown in far-red light than in those grown in darkness. After the 5 day stage the absorption at 636 nm relative to 650 nm decreased with age, and at the 8 day stage the spectra were almost the same in both types of leaves. Low temperature fluorescence spectra of the leaves also showed a change in the ratio between the different PChlide forms. The height of the fluorescence peak at 632 nm relative to the peak at 657 nm was higher in leaves grown in far-red light than in dark-grown leaves. – After exposure of the leaves to a light flash, the half time for the Shibata shift was measured. It increased with age both for leaves grown in darkness and in far-red light; but in older leaves grown in far-red light (7–8 days) the half time was slightly longer than in dark-grown leaves. – The chlorophyll accumulation in white light as well as the leaf unrolling were faster for leaves pre-irradiated with far-red light. The total length of the seedlings was equal or somewhat shorter in far-red light, but the length of the coleoptile was markedly reduced from 8.1 ± 0.1 cm for dark-grown seedlings to 5.2 ± 0.1 cm for seedlings grown in far-red light.     

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.