Red Irradiation Research Articles

Phenotypic Analysis of a Dwarf Wheat (Triticum aestivum L.) with Altered Phytochrome-Mediated Growth Responses

The influence of the amount of red light relative to far red light (red/far red ratio) on leaf-sheath elongation, leaf length, tillering, assimilate partitioning to shoots and roots, and chlorophyll content in a dwarf wheat (Triticum aestivum L. cv Tibet Dwarf) and seven other dwarf and standard cultivars was determined. All cultivars tested showed far red-stimulated leaf and leaf-sheath elongation except Tibet Dwarf. Tibet Dwarf was also unresponsive to a brief end-of-day exposure to far red light, although the other cultivars exhibited increased leaf-sheath and leaf length. In these cultivars, the effects of an end-of-day 5-min far red exposure were reversible by a subsequent 5-min red light exposure. Shoot/root ratios were higher and tillering was suppressed by increased far red irradiance in all cultivars except Tibet Dwarf. In addition, Tibet Dwarf was less responsive to dark-induced leaf and leaf-sheath elongation (etiolation) and retained chlorophyll in dark-adapted leaves longer than control cultivars. Tibet Dwarf did not differ from the other cultivars in leaf chlorophyll content. Western blot analysis, using an antibody against phytochrome A, showed that dark-grown Tibet Dwarf shoots contained at least twice the amount of detectable phytochrome A protein present in the other wheat cultivars. These results, plus the short, thick-stemmed, dark-pigmented phenotype of Tibet Dwarf suggest the possibility of a mutation in the phytochrome/signal transduction pathway.

PHYTOCHROME‐MEDIATED RAPID CHANGES IN THE LEVEL OF PHOSPHOINOSITIDES IN ETIOLATED LEAVES OF Zea mays

Abstract—The role of the active form of phytochrome in Zea mays on the polyphosphoinositide cycle was studied. As little as 15 s of red irradiation of etiolated leaves immediately increased the level of phosphatidylinositol bisphosphate (PIP2) 3–6‐fold compared to unirradiated leaves. The elevated level of PIP2 decreased with longer red irradiations up to 5 min, but remained higher than in unirradiated leaves. The level of PIP2 decreased if red irradiation was followed by far‐red irradiation. Far‐red alone had no effect. Levels of phosphatidylinositol phosphate (PIP) and phosphatidylinositol did not change significantly. Since red irradiation significantly changed PIP, but not PIP, photocontrol appears to be at the PIP kinase and phospholipase level. In related studies of the effect of light on phospholipids, 5 min of red irradiation induced significant decreases in phosphatidylcholine and phosphatidylethanola‐mine.

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.

Phytochrome‐mediated changes in the ATP content of Kalanchoë blossfeldiana seeds

ABSTRACTOne short red (R) irradiation increases the ATP content of Kalanchoë blossfeldiana Poelln. cv Feuerblüte seeds before onset of germination. Phytochrome control is demonstrated by the full R/far‐red light (FR) reversibility of the effect in water imbibed seeds. In seeds imbibed in the presence of gibberellin A3 (GA3, one short R exposure already increases the ATP content when given 2h after start of imbibition, showing phytochrome control at the energy‐metabolic level when one R pulse cannot yet induce germination. After longer imbibition periods in the presence of GA3, one short FR irradiation also increases the ATP content of ungerminated Kalanchoë seeds. The time course of the ATP levels after a R or FR germination inducing irradiation shows an initial increase that clearly preceeds germination. A second increase starts about 15 h after irradiation and is most probably the consequence of the germination itself. The results suggest that, in Kalanchoë seeds, the increase in ATP levels, induced by irradiation(s) and preceding germination, is a phytochrome‐mediated process, supplying energy, required for germination.

Circadian rhythm and fast responses to blue light of photosynthesis in Ectocarpus (Phaeophyta, Ectocarpales)

Photosynthesis of Ectocarpus siliculosus (Dillwyn) Lyngb. under continuous saturating red irradiation follows a circadian rhythm. Blue-light pulses rapidly stimulate photosynthesis with high effectiveness in the troughs of this rhythm but the effectiveness of such pulses is much lower at its peaks. In an attempt to understand how blue light and the rhythm affected photosynthesis, the effects of inorganic carbon on photosynthetic light saturation curves were studied under different irradiation conditions. The circadian rhythm of photosynthesis was apparent only at irradiances which were not limiting for photosynthesis. The same was found for blue-light-stimulated photosynthesis, although stimulation was observed also under very low red-light irradiances after a period of adaptation, provided that the inorganic-carbon concentration was not in excess. Double-reciprocal plots of light-saturated photosynthetic rates versus the concentration of total inorganic carbon (up to 10 mM total inorganic carbon) were linear and had a common constant for half-saturation (3.6 mM at pH 8) at both the troughs and the peaks of the rhythm and before and after blue-light pulses. Only at very low carbon concentrations was a clear deviation found from these lines for photosynthesis at the rhythm maxima (red and blue light), which indicated that the strong carbon limitation specifically affected photosynthesis at the peak phases of the rhythm. Very high inorganic carbon concentrations (20 mM) in the medium diminished the responses to blue light, although they did not fully abolish them. The kinetics of the stimulation indicate that the rate of photosynthesis is affected by two blue-light-dependent components with different time courses of induction and decay. The faster component seemed to be at least partially suppressed at red-light irradiances which were not saturating for photosynthesis. Lowering the pH of the medium had the same effects as an increase of the carbon concentration to levels of approx. 10 mM. This indicates that Ectocarpus takes up free CO2 only and not bicarbonate, although additional physiological mechanisms may enhance the availability of CO2.

Recovery of Growth of Soybean Hypocotyls under Continuous Red Light

Summary Red light accelerated the elongation of the hooks of dark-grown soybean seedlings ( Glycine max L.) but suppressed the elongation of the elongation zone of the hypocotyls. The lag time prior to the acceleration of hook elongation by red light was longer by about 19 h than that associated with the suppression of elongation of the elongation zone. Twenty-seven h after the onset of red light, the enhanced elongation of the hook part balanced the reduced elongation of the elongation zone, and the plant resumed stern elongation at the maximum rate in spite of continued red irradiation. Subsequently, the growth rate of soybean seedlings exposed continuously to red light was equal to that of non-irradiated seedlings of the same age.

Light regulates symplastic communication in etiolated corn seedlings

The effect of light on symplastic communication in dark grown maize seedlings was tested using the symplastic probe carboxyfluorescein. Prior white‐light irradiation stimulated longitudinal transport of carboxyfluorescein in the mesocotyl stele of dark‐grown seedlings. The stimulation was multiphasic with a positive followed by a negative phase. Lateral transport from the stele into the mesocotyl cortex was inhibited by the prior white‐light irradiation. The inhibitory effect of prior white‐light irradiation on lateral transport was completely photo‐modulatable by terminal farred and far‐red/red irradiations, suggesting the involvement of phytochrome. The stimulatory effect of the prior white irradiation observed for the stele, however, was not photoreversible. It is suggested that environmental factors, such as light, might modulate growth and development, in part, by modulating symplastic cell to cell communication and thus the distribution of nutrients and growth regulators.

Mechanism of the Inhibition of Cell Division of Plant Cells by Ionizing Radiations

Some plants are harmed by relatively low doses of ionizing radiations due to the selective radiosensitivity of certain vital cellular processes. For instance, the growth of barley (Hordeum vulgare L.) roots of germinating seeds is completely inhibited after a dose of 400 R of y radiation (1). Following irradiation, the root elongates normally to a finite length (about I cm) and then ceases to lengthen. This cessation of root growth is due to the stoppage of cell division in the root tip. The mechanism of this selective inhibition of cell division is not known. Chromosomal damage could be one possibility. However, some investigations that I have conducted suggest that there are other possibilities to consider. This letter summarizes these studies, and it is hoped that it will be of help to those interested in the problem. A little over 20 years ago, I reported that excised barley root tips from seedlings could be made to adhere to a negatively charged glass surface in a solution of several chemicals under red light (about 660 nm), and that subsequently they could be induced to detach from the glass surface under far-red light (about 730 nm) (2). The attachment is due to the generation of a positive electric charge in the outer membrane of root tip cells, and the detachment is due to its nullification. The entire photoprocess of attachmentdetachment can be completed in I to 2 min. This rapid photoreversibility can be sustained for several cycles. The photoreceptor for this process is a chromoprotein involved in growth processes named (3) with photoreversible properties. Following red irradiation, its maximum absorbancy at 660 nm shifts to 730 nm, and following far-red irradiation it shifts back to 660 nm. A few years later, I discovered that this photoreversible attachment of barley root tips is very sensitive to low doses of X rays. Loss of photoreversibility can be detected within a few minutes following exposure to a few hundred R of X rays (4). Since phytochrome is involved in growth processes, it is possible that the system damaged by ionizing radiation resulting in inhibition to cell division and the system involved in photoreversible attachment of root tips controlled by phytochrome could be one and the same. Moreover, the early detection of a radiation effect on root tip attachment suggests that the lesion could be the initial site of the effect of X irradiation. Immediately the possibility arises that the photoreceptor, phytochrome, could be dam ged by ionizing radiations. Any damage to it or its fun tion could result in deleterious consequences to growth. I have looked for changes in phytochrome absorbancy following X irradiation and have found none, indicating that the phytochrome molecule is probably not damaged. The inhibition to root growth through the selective stoppage of cell division can be seen in other circumstances. Notably, a deficiency of boron, an essential trace element in plant nutrition, results in termination of cell division in the root tip with little or no effect on root elongation. Similarly, low concentrations of chlorine selectively inhibit root growth through stoppage of cell division in root tips. Can it be possible that ionizing radiations, phytochrome, boron, and chlorine all act on the same system that controls cell division?

Poultry irradiation: For hygiene/safety and market‐life enhancement

Abstract The irradiation of fresh, raw poultry and of further‐processed poultry products has been the subject of research, testing, and evaluation since the late 1940s, along with red meat and other food irradiation applications. An enormous bank of information on all aspects of poultry irradiation has been developed over this period, through what has been a truly global effort. This review takes an up‐to‐date look at key aspects beginning with a look at why one might consider poultry irradiation in the first place. Since the international poultry trade sector has participated in poultry irradiation R&D in various ways over the years, and, since industrial‐scale poultry irradiation is now taking place in a few countries and has recently been approved in the United States, it is worthy of serious consideration by poultry producers everywhere. The ultimate business decision to adopt the technology is a complex one best addressed by a case‐by‐case industrial feasibility study. The ultimate personal decision ...

Red Light-mediated Two-dimensional Growth in Gametophytes of the Ferns Adiantum capillus-veneris L.and Dryopteris filix-mas (L.) Schott

Summary In contrast to what is known about growth of gametophytes of the ferns Adiantum capillus-veneris and Dryopteris filix-mas in red light, the results presented demonstrate that a certain percentage of gametophytes of both species can undergo a transition to biplanar growth under red irradiation whether this is given continuously or interrupted by a dark period of several weeks.Unlike blue light-grown prothallia, two-dimensional development in red light only occurs after the formation of a fairly long filamentous part.

THE PHOTOSYNTHETIC APPARATUS OF Acetabularia mediterranea GROWN UNDER RED OR BLUE LIGHT. BIOPHYSICAL QUANTIFICATION and CHARACTERIZATION OF PHOTOSYSTEM II and ITS CORE COMPONENTS

Abstract— The photosynthetic activity of white light‐grown Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) decreases under continuous red light to less than 20% within 3 weeks. Subsequent blue light reactivates photosynthesis within a relatively short period of 3 days. In a former publication (Wennicke and Schmid, Plant Physiol.84,1252–1256, 1987) we have shown that the regulated rate limiting step, which is an immediate light driven reaction, is part of photosystem II (PS II). The following biophysical properties of PS II were analyzed in thylakoids isolated from algae grown 3 weeks under either blue or red light with or without subsequent 3 days of blue light illumination: (a) fluorescence induction in the short time domain dominated by QA reduction, (b) the slow fluorescence decline reflecting pheophytin photoaccumulation, (c) absorption changes at 320 and 830 nm under repetitive flash excitation as indicator for the turnover of QA and P680, respectively, (d) oscillation pattern of the oxygen yield by a flash train in dark adapted samples and (e) the binding capacity for atrazine. None of these PS II functions were severely affected, but a minor impairment of20–30% was observed in the thylakoids from algae grown for 3 weeks in red irradiation. The changes do not fully account for the drastic reduction of the electron transport through PS II which was 80% after red light treatment. Therefore, the regulated rate‐limiting step appears to not be mainly located in the PS II core complex itself. It seems likely that the regulation process predominantly comprises the antenna system.

Long-Lasting Light Effects in Imbibed Kalanchoë blossfeldiana Seeds in the Presence of Gibberellic Acid

Two brief red (R) irradiations, separated by 24 hours, given to Kalanchoë blossfeldiana Poelln. cv Feuerblüte seeds, made secondarily dormant by a prolonged dark incubation period on water and transferred to GA(3), induce very low germination. Some effect of these irradiations is preserved, however, during a long dark interval in fully imbibed seeds and greatly increases the germination induced by another brief R exposure. This long-lasting light effect is, at 20 degrees C, only lost after a dark interval of about 1 month. It can also be induced by two brief far-red (FR) exposures. Its preservation is temperature-dependent, low temperatures being favorable. Light-induced changes in the ATP-content were demonstrated during preservation and expression of the long-lasting light effect, indicating a long-lasting metabolic change. In seeds with primary dormancy sown on GA(3), an analogous long-lasting light effect is induced by one or two brief R or FR irradiations, even when they are given before germination can take place. The presence of GA(3), which was shown to induce a very low fluence germination response in Kalanchoë seeds, is required for the occurrence of the long-lasting light effect. The data suggest long-term preservation of some effect(s) of Pfr rather than persistent presence of Pfr itself.

Role of red light in hair-whorl formation induced by blue light in Acetabularia mediterranea

After a pre-treatment with red light, hair formation at the growing tip of the siphonaceous green alga Acetabularia mediterranea Lamour. (= A. acetabulum (L.) Silva) can be induced by a pulse of blue light. Red light is needed again after the inductive blue-light pulse if the new whorl of hairs is to develop within the next 24 h. In order to investigate the role of this red light, the duration of the red irradiation was varied and combined with periods of darkness. The response of hair-whorl formation was dependent on the total amount of red light, regardless of whether the red irradiation followed the blue pulse immediately or was separated from it by a period of darkness. Furthermore, periods of exposure to the photosynthesis inhibitor 3-(3',4'-dichlorophenyl)-1-1dimethylurea had a similar effect to darkness. Both observations indicate that this red irradiation acts as a light source for photosynthesis. Whether or not the red light had an additional effect via phytochrome was tested in another type of experiment. The dependence of hair-whorl formation on red-light irradiance in the presence of simultaneous far-red irradiation was determined for the pre-irradiation period as well as for the irradiation period after the blue pulse. In both experiments, far-red light caused a small promotion of hair-whorl formation when low irradiances of red light were used. However, these differences were attributable to a low level of photosynthetic activity (which in fact was measurable) caused by red light reflected in the growth chamber. Furthermore, lowering the proportion of active phytochrome by far-red light would be expected to suppress hair-whorl formation. The influence of far-red light was also tested in a strain of Acetabularia mediterranea that developed hair whorls in about 20% of cells even when kept in complete darkness after the blue-light pulse. Far-red irradiation had no effect. These results strongly indicate that phytochrome is not involved in hair-whorl formation. Rather it is concluded that the effects of red light are caused by photosynthesis.

Morphogenic Responses to Propagule Trimming, Spectral Irradiance, and Photoperiod of Grapevine Shoots Recultured in Vitro

Abstract In an effort to improve grapevine micropropagation, shoot proliferation and rooting in response to explant preparation and incubation conditions were investigated. The explants were three- to four-node shoots (15 mm) of the Vitis hybrid ‘Remaily Seedless’ cultured in vitro. The factors considered were white light of diverse spectral irradiance distributions, blue light, red light, 10-hr light/14-hr dark and 16-hr light/8-hr dark photoperiods, and leaf and/or apex removal from the explant. In white light and short days, leaf and/or apex removal did not affect shoot production; however, more shoots of at least three nodes, the desired size for micropropagation, were produced when leaves were retained. Shoot production was significantly greater in blue than in red light. Differences in shoot production in white lights of various spectral irradiance distributions were attributed to differences in blue : red irradiance ratios. Shoot production was greater with ratios of 0.6 to 0.9 than with lower and higher ratios. A greater percentage of subcultured shoots rooted in long days and red light than in blue or white light. The number of roots produced per shoot, and total root length, were also greater in red light.

THE CONTROL OF METABOLISM BY BLUE LIGHT IN Acetabularia mediterranea—I. LIGHT DEPENDENCY AND EVIDENCE FOR POST‐TRANSCRIPTIONAL CONTROL OF UDPG PYROPHOSPHORYLASE AND PYRUVATE KINASE

Abstract— After a preculture in white light, Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) was irradiated with either continuous red or blue light. In crude extracts, the specific activities of UDPG pyrophosphorylase(uridine–5′‐triphosphate: glucose 1‐phosphate uridylyltransfer‐ase EC 2.7.7.9) and pyruvate kinase(adenosine–5′‐triphosphate: pyruvate phosphotransferase EC 2.7.1.40) varied as a result of the different light qualities. In red light activities decreased to 40% and 60%, respectively, within 3 weeks of irradiation, while under blue light, if not preceded by a red irradiation, the activities remained unchanged. After prolonged red light, high enzyme activities were restored by blue light with a relatively rapid time course (3 to 4 days for completion) compared with the decrease in red light. A fluence‐response curve for the activation of UDPG pyrophosphorylase showed two phases with different sensitivities for blue light.The blue light‐mediated increase in the activities of both enzymes was unaffected when the cell nucleus was removed before the beginning of the blue irradiation. The increase was inhibited by cycloheximide but not by chloramphenicol. Thus, functional cytosolic translation but not de novo transcription products are presumed to be required for the increase in specific enzyme activities in vivo.

On the Mechanism of Skotodormancy Induction in Grand Rapids Lettuce ( Lactuca sativa L.) Seeds

Summary Prolonged imbibition of lettuce ( Lactuca sativa L. cv. Grand Rapids) seeds in darkness results in a gradual loss of responsiveness to a short Red irradiation. This induction of skotodormancy was clearly shown to be temperature dependent above a critical temperature between 15 and 17.5 °C. At lower temperatures (15 and 10 °C) skotodormancy was not imposed at all, at least during the 20-day incubation period tested. When lettuce seeds were kept under continuous Far Red, instead of being pretreated in darkness, the induction of dormancy did not attain the final level of the dark-incubated seeds. Moreover, the responsivity to R was eventually resumed after several days in cFR and the higher the fluence rate the earlier the recovery of responsivity. In fact, at the highest FR fluence rate used, no additional dormancy was imposed. However, in comparison to the untreated seeds, a marked delay of germination was observed for the FR pretreated ones. Both the induction of skotodormancy and the reverse effect produced by high fluence cFR are discussed in terms of phytochrome and its postulated reaction partner X.

Influence of red irradiation and of removal of integuments on the metabolism of (±) [2‐14C]‐abscisic acid by Lactuca sativa Grand Rapids achenes

Achenes of Lactuca saliva L. cv. Grand Rapids, imbibed for 6 h in water or in a 10 µM solution of non‐radioactive abscisic acid (ABA), were cultivated on (2‐14C]‐ABA (10 µM) for 40 to 90 h. Red irradiation (660 ± 2.5 nm, 5 min, 2 W m ‐2) or removal of integuments were carried out before transfer to (2‐14C]‐ABA. When both treatments were applied, irradiation preceded removal of integuments. Imbibition and culture took place in darkness at 24°C. Two acidic diethyl ether phases, which contained the free acids (free phase) and the acids released after mild alkaline hydrolysis, respectively, were isolated. They were analyzed by thin layer chromatography (TLC). as well as the remaining aqueous phase.Both red irradiation and removal of integuments led to increased [2‐14C|‐ABA uptake. Application of ABA during imbibition partly limited the stimulating effect of red irradiation on radioactive ABA uptake. Red irradiation stimulated [2‐14C|‐ABA metabolism by achenes, favouring the formation of the polar compound found in the remaining aqueous phase. Removal of the integuments stimulated metabolism notably, leading to an increase of the radioactivity in the remaining aqueous phase. This treatment also induced the appearance of new metabolites in the free phase (compound believed to be 7'‐hydroxy‐ABA) as well as in the remaining aqueous phase. The glucose ester of ABA was the only representative compound of the ester phase. Irrespective of the experimental conditions, there was no classical oxidative metabolism indicating that oxygen was not the limiting factor.

Phytochrome Photoconversion in Vivo

The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons.

The effect of some growth regulators on light‐induced germination of Paulownia tomentosa seeds

Grubišié, D., Konjevié, R. and Neškovié, M. 1988. The effect of some growth regulators on light‐induced germination of Paulownia tomentosa seeds. ‐ Physiol. Plant. 72: 525–528.The germination of Paulownia tomentosa Steud. (Empress tree) seeds can be induced either by red light (R) or by exogenously applied gibberellic acid (GA3). The R induced germination is completely suppressed by far red irradiation, abscisic acid (ABA) or by growth retardants such as ancymidol, tetcyclacis and paclobutrazol, though not by AMO 1618 or chloro‐choline chloride (CCC). The inhibition caused by far red light (FR) and growth retardants can be overcome by GA3, while the ABA‐induced inhibition can be reversed by fusicoccin (FC), which does not reverse the inhibition caused by FR or growth retardants. It is noteworthy that the germination of light insensitive wheat, corn, alfalfa and mung bean seeds is not inhibited by growth retardants.

Phytochrome Regulation of Greening in Barley—Effects on Chlorophyll Accumulation

Red light treatment of dark-grown 6-day-old barley seedlings (Hordeum vulgare L.) strongly reduces the lag in chlorophyll accumulation in subsequent white light over that found in dark control seedlings placed under white light. Fluence-response studies show that the effect has both very low fluence and low fluence components. Kinetic studies indicate that the reduction in lag begins immediately following either a low fluence or a very low fluence red irradiation, with the initial rate of change significantly lower after the very low fluence treatment and showing sharp far red-absorbing form of phytochrome dependence. In both cases, the effect is maximal after roughly 4 hours, either remaining fairly constant (very low fluence) or declining somewhat (low fluence) thereafter. Saturating far red light alone yields a response equivalent to very low fluence red, and will reverse only the low fluence component of the red response. Escape from far red reversibility occurs gradually over about a 3 hour period. Since the kinetics described here differ from those in the literature related to phytochrome effects on transcription of the mRNA for the light-harvesting chlorophyll a/b-binding protein, we conclude that the phytochrome-regulated component of chlorophyll accumulation is not limited by transcription of the mRNA for its major apoprotein. Leaf segments vacuum-infiltrated with water retain the capacity to green in white light. If they are infiltrated with mannitol solutions of various concentrations, their capacity to green declines sharply at concentrations above 0.2 molar. These results bear on interpretation of run-on transcription experiments with isolated nuclei: preparation of the nuclei involves enzymic digestion of the tissue in the presence of 0.7 molar mannitol for 2.5 hours, to obtain protoplasts prior to breaking the cells. The results here make it unlikely that normal transcriptional regulation is occurring during this procedure.