Photocontrol of Anthocyanin Synthesis: VI. Spectral Sensitivity, Irradiance Dependence, and Reciprocity Relationships

The spectral sensitivity and the irradiance dependence of anthocyanin synthesis, a “high irradiance response,” in cabbage (Brassica oleracea, cv. Red Acre) and tomato (Lycopersicon esculentum, cv. Beefsteak) seedlings exposed to continuous irradiation depend upon the length of the exposure. In cabbage, blue and red are more effective than far red when the irradiations are shorter than 12 hours and less effective than far red when the irradiations are longer than 12 hours. The irradiance dependence is negligible under red and becomes evident under blue and far red red only for exposures longer than 12 hours. Anthocyanin synthesis under intermittent light treatments, of efficiency comparable to that of continuous treatments, obeys the Bunsen-Roscoe reciprocity law and is a function of the dose (irradiance × time), rather than of the irradiance alone. The validity of the reciprocity relationships suggests that only one photoreceptor is responsible for the photocontrol of the response in the blue, red, and far red spectral regions. The characteristics of the response suggest that the photoreceptor is phytochrome, at least in cabbage.     

An Analysis of Phytochrome-mediated Anthocyanin Synthesis

Phytochrome (far red form) alone can mediate anthocyanin synthesis in the mustard seedling (Sinapis alba L.). Complete photoreversibility and reciprocity, for both red and far red light exposures over a period of at least 5 minutes, demonstrate this phytochrome involvement.     

Photocontrol of Anthocyanin Synthesis: III. The Action of Streptomycin on the Synthesis of Chlorophyll and Anthocyanin

Streptomycin enhances the synthesis of anthocyanins and inhibits the synthesis of chlorophylls and the development of chloroplasts in dark-grown seedlings of cabbage (Brassica oleracea), mustard (Sinapis alba), tomato (Lycopersicon esculentum), and turnip (Brassica rapa) exposed to prolonged periods of irradiation in various spectral regions. These results suggest that the contribution of photosynthesis to light-dependent high irradiance reaction anthocyanin synthesis in seedlings of cabbage, mustard, tomato, and turnip is minimal, if any at all. So far, phytochrome is the only photoreceptor whose action in the control of light-dependent anthocyanin synthesis in seedlings of cabbage, mustard, tomato, and turnip has been satisfactorily demonstrated.     

Photocontrol of Anthocyanin Synthesis: VII. Factors Affecting the Spectral Sensitivity of Anthocyanin Synthesis in Young Seedlings

Light-dependent anthocyanin synthesis is a typical high irradiance response (HIR) of plant photomorphogenesis. The spectral sensitivity of this response in young seedlings of cabbage and tomato is strongly affected by the length and mode of application of the light treatments. This observation suggests that the different experimental conditions, used in different action spectroscopy studies, might have been responsible, at least in part, for some of the reported differences in the characteristics of the HIR action spectra of different response-system combinations. In both cabbage and tomato, the values of the far red/blue, far red/red, and blue/red action ratios increase with increasing durations of the light treatments; this finding is in agreement with hypotheses of K. M. Hartmann (1966, 1967) and E. Schäfer (1975) for phytochrome action in the HIR. The similarity in the trend of change of the values of the action ratios suggests the possibility that the photomorphogenic pigment system, involved in the photoregulation of anthocyanin synthesis, may be the same in cabbage and tomato, even though there are some differences in the spectral sensitivity of the response between the two species.     

Photomanipulation of phytochrome in lettuce seeds

Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze-dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red-absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant.     

Changes in Phytochrome Expressed by Germination of Amaranthus retroflexus L. Seeds

Effects of red (600 to 680 nanometers) and far red (700 to 760 nanometers) irradiances on Amaranthus retroflexus L. seeds indicate that synthesis of phytochrome in the red-absorbing form takes place in water-imbibed nongerminating seeds at 35 C. After 96 hours in darkness, conversion of about 0.10% phytochrome to the far red-absorbing form induces 50% germination. Continuous far red radiation at 35 C with an irradiance of 0.4 × 10⁻¹⁰ Einsteins per square centimeter per second caused photoinactivation of phytochrome about equal to the rate of synthesis. Germination of seeds at 35 C, following far red irradiation adequate to establish the photostationary state, is enhanced by holding at 26 C for 16 minutes. Germination is unaffected relative to controls at constant temperature, if the period at 26 C precedes irradiation. The results indicate a quick response to action of phytochrome in a germination process.     

Turnover of phytochrome in pumpkin cotyledons

By using density labeling, it was found that the protein moiety of phytochrome is synthesized de novo in the red-absorbing form in cotyledons of dark-grown pumpkin (Cucurbita pepo L.) seedlings, as well as those irradiated with red light and returned to the dark. The rate of synthesis appears to be unaffected by the light treatment. Turnover of the red-absorbing form was also detected in dark grown seedlings using density labeling, while turnover of the far red-absorbing form is already implied from the well known “destruction” observed in irradiated seedlings. In both cases, true degradation of the protein is involved, but the rate constant of degradation of the far red-absorbing form may be up to two orders of magnitude greater than that of the red-absorbing form. The data indicate that, in pumpkin cotyledons, phytochrome levels are regulated against a background of continuous synthesis through divergent rate constants of degradation of the red and far red-absorbing forms and the relative proportions of the two forms present.     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Phytochrome Control of Germination of Rumex crispus L. Seeds Induced by Temperature Shifts

High germination of curly dock (Rumex crispus L.) seeds is evident after suitable imbibition and temperature shift treatment, but germination at constant temperatures fails without an input of far red-absorbing form of phytochrome. Preliminary imbibitions at high temperatures (30 C) sharply reduce germination induced by temperature shifts. High germination may be restored by low energies of red radiation, or by brief far red adequate for the photosteady state. Prolonged far red during imbibition also nullifies temperature shift-induced germination. After prolonged far red, high germination may be restored by red radiation of an energy dependent upon the duration of the far red treatment. The evidence supports the conclusion that dark germination induced by temperature shifts arises from the interaction of pre-existent far red-absorbing form of phytochrome in the mature seeds with the temperature shift.     

Phytochrome Transformation and Action in Seeds of Rumex crispus L. during Secondary Dormancy

Promotion of germination by red light fails after prolonged dark imbibition of Rumex crispus L. seeds, indicative of a secondary dormancy. The degree and rate of inception of the dormancy increases with increasing temperature. Following establishment of the dormancy, germination response to red light can be restored by either prolonged cold treatment or brief high temperature shifts. Loss of phytochrome was not a factor in the initial establishment of the dormancy. When the seeds are in secondary dormancy, the chromophore of phytochrome can be transformed to the far red-absorbing form, but the far red-absorbing form cannot induce germination. The responses to changes in temperature suggested dependence of germination on order disorder transitions in components of the seeds.     

Rhizoid Differentiation in Spirogyra: III. Intracellular Localization of Phytochrome

Localization of phytochrome which mediates rhizoid differentiation in Spirogyra was investigated. The red-absorbing form of phytochrome (Pr) seems to be distributed all over the cell periphery which remained in the centripetal end part after the centrifugation, as rhizoids formed equally well with red spotlight irradiation of three different parts of an end cell, i.e. distal end, middle, and proximal end, and with irradiation of centrifugal and centripetal end parts of a centrifuged end cell. The Pr distribution was confirmed with an experiment using far red irradiation over the entire cell, centrifugation, and red spotlight irradiation. The Pr-phytochrome molecules appeared to be mobile because no dichroic orientation was shown with polarized red spotlight irradiation. On the contrary, it is suggested that far red-absorbing form of phytochrome molecules are evacuated from the centripetal end part by the centrifugation in an experiment involving red irradiation over the entire cell-centrifugation-far red spot irradiation. Rhizoid formation was repressed markedly by far red irradiation of the centrifugal end part but not of the centripetal end part.     

Light Control of Anthocyanin Biosynthesis in Zea Seedlings

Evidence for involvement of two non-photosynthetic pigments in photoinduction of anthocyanin biosynthesis in the roots and mesocotyls of Zea mays L. seedlings is presented. Short (5 min), low energy (4.5 × 10³ J m^?2) fluences of red light neither induced anthocyanin synthesis nor enhanced phenylalanine ammonia-lyase activity in dark-grown maize seedlings. Little anthocyanin synthesis and no enhancement of phenylalanine ammonia-lyase activity was induced by continuous far-red light. Continuous white or blue light induced both anthocyanin synthesis and enhanced phenylalanine ammonia-lyase activity. These results show that phytochrome alone cannot induce anthocyanin synthesis in maize seedlings. However, a strong phytochrome mediation of white light induced pigment synthesis was demonstrated. This effect was not demonstrable with white light enhanced phenylalanine ammonia-lyase activity, indicating that phytochrome controls another step in anthocyanin biosynthesis.     

Photoregulation of Anthocyanin Synthesis : VIII. Effect of Light Pretreatments

A comparative study of the spectral sensitivity of anthocyanin production in dark-grown and light-pretreated systems was carried out in Brassica oleracea L., Lycopersicon esculentum Mill., Secale cereale L. and Spirodela polyrrhiza L. Light pretreatments bring about an enhancement of the inductive, red-far red reversible response in all systems, a decrease of the continuous irradiation response in cabbage, rye, and tomato seedlings, and an enhancement of the continuous irradiation response in cabbage leaf disks. Light pretreatments also bring about a marked change in the spectral sensitivity of the continuous irradiation response. The different effect of light pretreatments on the photosensitivity of the response to short and long wavelength irradiations suggests that two photoreceptors, phytochrome and cryptochrome, may be involved in the photoregulation of anthocyanin production.     

Genetic Regulation of Development in Sorghum bicolor: VI. The ma(3) Allele Results in Abnormal Phytochrome Physiology

Physiological processes controlled by phytochrome were examined in three near-isogenic genotypes of Sorghum bicolor, differing at the allele of the third maturity gene locus. Seedlings of 58M (ma₃^R ma₃^R) did not show phytochrome control of anthocyanin synthesis. In contrast, seedlings of 90M (ma₃ma₃) and 100M (Ma₃Ma₃) demonstrated reduced anthocyanin synthesis after treatment with far red and reversal of the far red effect by red. De-etiolation of 48-hour-old 90M and 100M dark-grown seedlings occurred with 48 hours of continuous red. Dark-grown 58M seedlings did not de-etiolate with continuous red treatment. Treatment of seedlings with gibberellic acid or tetcyclacis, a gibberellin synthesis inhibitor, did not alter anthocyanin synthesis. Levels of chlorophyll and anthocyanin were lower in light-grown 58M seedlings than in 90M and 100M. Etiolated seedlings of all three genotypes have similar amounts of photoreversible phytochrome. Crude protein extracts from etiolated seedlings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phytochrome was visualized with Pea-25, a monoclonal antibody directed to phytochrome from etiolated peas. The samples from all three genotypes contained approximately equivalent amounts of a prominent, immunostaining band at 126 kD. However, the sample from 58M did not show a fainter, secondary band at 123 kD that was present in 90M and 100M. The identity and importance of this secondary band at 123 kD is unknown. We propose that 58M is a phytochrome-related mutant that contains normal amounts of photoreversible phytochrome and normal phytochrome protein when grown in the dark.     

Dark Reversion of Phytochrome in Lettuce Seeds Stored in a Water-saturated Atmosphere

Dark reversion of the far red-absorbing form of phytochrome, which does not occur in dry lettuce (Lactuca sativa var. Grand Rapids) seeds, appears to take place in seeds stored in a water-saturated atmosphere. The water content (approximately 70% after 10 days) of such seeds is insufficient to support germination; however the treatment enhances germination in seeds stored for 1 to 5 days, but this enhancement subsequently disappears, and the effect of extended storage (up to 28 days) is inhibiting. The half-time for dark far red-absorbing phytochrome reversion is 7 to 8 days, and at this time it can be completely reversed by exposing the seeds to a flash of red light. Storage of more than 7 to 8 days decreases red light enhancement of germination.     

Photoregulation of Anthocyanin Synthesis in Apple Fruit under UV-B and Red Light

The involvement of photomorphogenic photoreceptors in anthocyaninsynthesis was investigated in apple fruits under UV light from280 to 320 nm (UV-B) and red light (R). Short-term R treatmentwas ineffective in the induction of anthocyanin synthesis butthe involvement of phytochrome was indicated by the resultsof long-term irradiation (18 h) with R. The inductive effectof 18 h UV-B on anthocyanin synthesis was stimulated synergisticallyby subsequent irradiation with R for 15 min, and the R, far-redlight (FR) photorevesibility of this effect indicated the involvementof phytochrome in this synergism. The effect of UV-B on anthocyaninsynthesis was not influenced by subsequent irradiation withFR, suggesting that the effect of UV-B was independent of phytochrome,and that a specific photoreceptor for UV-B was involved. WhenR was given simultaneously with UV-B (18 h), anthocyanin wassynthesized at a much higher rate than it was after sequentialirradiation with UV-B and R. Photosynthesis was shown to beinvolved inthis synergistic increase in the synthesis of anthocyanin,although the involvement of phytochrome in the expression ofthis response, at least in part, was suggested by a reductionin the rate of anthocyanin synthesis by FR. (Received March 14, 1988; Accepted September 28, 1988)     

Photomorphogenesis in Arabidopsis thaliana (L.) Heynh: Threshold Intensities and Blue-Far-red Synergism in Floral Induction

Arabidopsis seeds were germinated on sterile mineral agar supplemented with 1% glucose and cultured under continuous light regimes. With 4-hour incandescent plus 20-hour monochromatic illumination in the region from 400 to 485 nanometers there was effective floral induction at an intensity of 100 microwatts per square centimeter. Exclusion of far red wave lengths from the 4-hour incandescent period sharply reduced the effectiveness of subsequent monochromatic blue light in promoting floral induction. Delayed floral induction occurred under continuous incandescent light lacking far red and was attributable to the blue wave lengths. Continuous 485 nanometer (100 microwatts per square centimeter) exposure without any white light treatment during the postgermination growth period was ineffective in floral induction and meristem development. Light at 730 nanometers under the same conditions was partially effective, whereas energy between 500 and 700 nanometers was completely ineffective. When continuous monochromatic light at a 3-fold higher energy level was administered, all photomorphogenic responses were accomplished with 485 nanometer light, including germination and 100% floral induction without any white light treatment at any time during the experiment. Almost equal quantum effectiveness was calculated when equivalent quantum flux densities in the region from 710 to 740 nanometers or at 485 nanometers were used. It is postulated that floral induction in Arabidopsis may be the result of a continuous excitation of a stable form of far red-absorbing phytochrome localized in or on a membrane, and that excitation can be either by direct absorption of energy by far red-absorbing phytochrome or by transfer from an accessory pigment.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

Red and far red effects on phenylalanine ammonia-lyase in raphanus and sinapis seedlings do not correlate with phytochrome spectrophotometry

In seedlings of Raphanus sativus (radish) and Sinapis alba (mustard), irradiation for 6 hours with far red light significantly increases the extractable activity of phenylalanine ammonia-lyase by the end of the light period. A schedule of 10 minutes red light-110 minutes darkness-10 minutes red-110 minutes darkness-10 minutes red-110 minutes darkness has no effect as compared to dark controls. However, the red light program maintains a level of far red-absorbing phytochrome always measurable by in vivo spectrophotometry during the 6-hour experimental period. We conclude that the far red effect on this enzyme and for this specific material cannot be explained solely by formation and maintenance of far red-absorbing phytochrome.     

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)