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 GA₃, 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°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 GA₃, 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 GA₃, 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.     

Analysis of the Effect of Light and Temperature on the Fluence Response Curves for Germination of Rumex obtusifolius

Both red light (10 minutes) and 35°C treatment (60 minutes) stimulate the germination of seeds of Rumex obtusifolius otherwise maintained in darkness at 25°C. Fluence response curves were determined for the effect of red light to stimulate germination of seeds with and without 35°C treatment. The endogenous far-red absorbing form (Pfr) level in the seeds was determined using short saturating fluences of wavelengths of light which maintain different proportions of phytochrome as Pfr at equilibrium. In the seed batches investigated, the endogenous Pfr level was found to be 4% or less of the total phytochrome. High dark germination after 35°C treatment does not result from an increase in sensitivity of the whole population to Pfr. Calculated fluence response curves for germination which best fit the experimental data suggest that seeds germinate in darkness after 35°C treatment because of a nonphytochrome-related process (overriding factor).     

Prevention of Action of Far-Red-Absorbing Phytochrome in Rumex crispus L. Seeds by Ethanol

Phytochrome-enhanced germination of curled dock (Rumex crispus L.) seeds is further stimulated by pretreatments in solutions of 0.5 to 2 molar methanol and 0.03 to ≥ 0.3 molar 2-propanol during a 2-day 20°C imbibition. Similar pretreatments in 0.1 molar ethanol, acetaldehyde, and n-propanol inhibit phytochrome-enhanced germination. If exposure to ethanol is delayed until 16 hours after a red irradiation, seeds escape the ethanol inhibition indicating a mechanism other than toxicity. The rate of escape from ethanol inhibition roughly parallels the escape from phytochrome control in seeds held in water only, indicating possible ethanol effects on phytochrome. It was found that ethanol pretreatment prevents the far-red absorbing form of phytochrome (Pfr) from acting but does not accelerate dark decay or prevent transformation. Ethanol inhibition may be prevented if ethanol pretreatment is at 10°C instead of 20°C, or may be overcome by transferring ethanol-pretreated seeds to 10°C in water. Similarly, ethanol inhibition can be overcome by a 2-hour 40°C temperature shift concluding the pretreatment. It is proposed that the ethanol causes perturbations at a membrane which prevent Pfr from acting.     

Effects of long-term storage on phytochrome-mediated germination in lettuce seeds

The effects of long-term seed storage on the physiological properties of phytochrome-mediated germination including water uptake, the temperature and light flunnce dependencies of germination and dark germination were studied. The fluenceresponse relationships of the brief irradiation with monochromatic red (660 nm, 7.5 W m⁻²) and far-red (750 nm, 6.6 W m⁻²) light at various times after sowing were also studied. The samples used consisted of three lots of seeds ofLactuca sativa L. cv. MSU-16, which had been harvested in 1976, 1979 and 1985 and stored dry for 9, 6 and 0 years, respectively, in darkness at 23±2 C until the experiments were carried out in July–August, 1985. Seeds with the longer storage periods showed the higher ability to germinate in both continuous darkness and continuous white fluorescent light at 20–30 C. In the seeds stored for 6 or 9 years, red light irradiation for 20 sec given at 15 min or more after sowing at 25 C induced as high a percent germination (85–95%) as those under continuous white fluorescent light. In the freshly harvested seeds, however, germination under continuous white fluorescent light (46%) was considerably lower than the germination induced by the red pulse (97%). Germination of the seeds decreased when the intervals between sowing and a far-red irradiation for 20 sec increased up to 100 min (or 30 min in the freshly harvested seeds). The far-red pulse given later than 100 min (or 6 hr in the freshly harvested seeds) after sowing resulted in an increased germination up to the dark-germination levels with increasing intervals between sowing and the pulse irradiation. Before or at 3 min after sowing, the seeds stored for 6 or 9 years were responsive to the far-red pulse although they were not or hardly responsive to the red pulse, while the freshly harvested seeds were responsive to both the far-red and the red pulses. These data indicate that normal functions of phytochrome completely survived in the dry seeds during storage at 25 C for as long as 6 or 9 years and that these functions are restored into full operation by means of imbibition. The differences in the dependence of germination on the time and fluence of a single pulse of red or far-red light seems to be related to the smaller water content throughout the imbibition in the seeds with the longer storage periods. The greater ability to germinate in the dark indicates the greater amounts of P_FR or the greater responsivity to P_FR, in the seeds with the longer storage periods.     

Dual action of respiratory inhibitors: inhibition of germination and prevention of dormancy induction in lettuce seeds

`Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15°C but poorly at 25°C in darkness. When held in dark at 25°C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15°C in darkness. Induction of dormancy at 25°C was prevented by exposure to CN<sup>−</sup>, azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N<sub>2</sub> as determined by subsequent germination at 15°C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN<sup>−</sup>, SHAM, and azide promoted dark germination at 25°C while at high levels they were inhibitory. Uptake of O<sub>2</sub> by seeds held at 25°C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O<sub>2</sub> uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and `alternative pathway' is effectively engaged in presence of CN⁻. The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors.

A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN⁻ (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN⁻, CN⁻ + SHAM) presumably leads to accumulation of `reducing power' with consequent improvement in O₂ uptake and oxidation rates of processes controlling germination or dormancy induction upon removal of the inhibitors.

     

Photoinduced Seed Germination of Oenothera biennis L: II. Analysis of the Photoinduction Period

The photoinduction period of Oenothera biennis L. seed germination was analyzed by varying the photoinduction temperature and by substituting red light pulses for continuous red light. At 24°C, seeds require 36 hours of continuous red light for maximal percent germination. The optimal photoinduction temperature is 32°C, with higher and lower temperatures being strongly inhibitory. A 30 minute exposure to far-red light, given immediately after a red light period of 1 to 36 hours, reduces germination by about 25%. Seeds escape from far-red inhibition with a half-time of 5 to 10 hours, depending on the length of the red exposure that precedes the far-red light. Periodic 15 minute pulses of red light can substitute for continuous red light in stimulating germination. Ted red light pulses, with 6 hours of darkness between successive pulses, cause maximal germination. The response to periodic red light is fully reversible by far-red light. Probit analysis of the periodic light response shows that as the length of the dark periods between successive pulses increases, less incident light is needed to induce germination but the population variance in light sensitivity remains constant. Probit analysis of the temperature response shows that as the photoinduction temperature increases from 16 to 32°C, less incident light is needed to induce germination and the population variance in light sensitivity also increases.     

Photoinduced Seed Germination of Oenothera biennis L: III. Analysis of the Postinduction Period by Means of Temperature

The postinduction period of Oenothera biennis L. seed germination was examined by temperature treatments. For all experiments, seeds received a standard 24 hour/24°C preinduction period and 12 hour/32°C photoinduction period. Germination is inhibited by postinduction temperatures above 32°C. When seeds are briefly incubated at 44°C and then transferred to 28°C, they germinate at a much lower percentage than 28°C controls. When thermally inhibited seeds are placed in the dark at 28°C for 20 hours, they can be promoted to germinate by a single pulse of red light. Seeds incubated at 12°C or below immediately after photoinduction enter a lag period in which they germinate slowly or not at all for a long time and then resume germination. The length of the lag period is exponentially related to the postinduction temperature. When seeds are incubated at a low temperature and then transferred to a warm temperature, they germinate much more rapidly than seeds not incubated at a low temperature. A model is proposed which is consistent with these and additional results. In the model, a germination promoter is irreversibly formed from a precursor and the synthesis of the precursor is favored at low temperatures and its degradation is favored at high temperatures.     

Photoinduced Seed Germination of Oenothera biennis L: I. General Characteristics

General characteristics of light-induced germination of Oenothera biennis L. seeds were investigated at 24°C. During dark imbibition, seeds reached maximal respiration in 7 hours and maximal water content and photosensitivity in 24 hours. After dark imbibition of 24 hours, seeds required a long exposure (>36 hours) to red or white light for maximal germination. Two photoperiods (12 and 2 hours) separated by a period of darkness of 10 to 16 hours gave near maximal germination. For the two photoperiod regime, the first light potentiates a reversible phytochrome response by the second light. A 35°C treatment for 2 to 3 hours in the dark immediately prior or subsequent to 8 hours of light caused a higher percentage of germination. A 2 hour treatment at 35°C also potentiates a reversible phytochrome response. Halved seeds germinated at 100% in light or darkness indicating that the light requirement of the seeds is lost in the halving procedure. After-ripened seeds required less light and germinated more rapidly and at higher percentages than seeds tested shortly after maturation.     

Mechanism of seed priming in circumventing thermodormancy in lettuce

Lettuce (Lactuca sativa L. cv Minetto) seeds were primed in aerated solutions of 1% K₃PO₄ or water at 15°C in the dark for various periods of time to determine the manner by which seed priming bypasses thermodormancy. Seeds which were not primed did not germinate at 35°C, whereas those which were primed for 20 h in 1% K₃PO₄ or distilled H₂O had up to 86% germination. The rate of water uptake and respiration during priming were similar regardless of soak solution. Cell elongation occurred in both water and 1% K₃PO₄, 4 to 6 h prior to cell division. Both processes commenced sooner in water than K₃PO₄. Radicle protrusion (germination) occurred in the priming solution at 21 h in water and 27 h in 1% K₃PO₄.

Respiration, radicle protrusion and cell division consistently occurred sooner in primed (redried) seeds compared to nonprimed seeds when they were imbibed at 25°C. Cell division and elongation commenced after 10 h imbibition in primed (redried) seeds imbibed at 35°C. Neither process occurred in nonprimed seeds. Respiratory rates were higher in both primed and nonprimed seeds imbibed at 35°C compared to those imbibed at 25°C, although radicle protrusion did not occur in nonprimed seeds which were imbibed at 35°C. It is apparent that cell elongation and division are inhibited during high temperature imbibition in nonprimed lettuce seeds. Seed priming appears to lead to the irreversible initiation of cell elongation, thus overcoming thermodormancy.

     

Phytochrome and Seed Germination. I. Temperature Dependence and Relative P(FR) Levels in the Germination of Dark-germinating Tomato Seeds

Germination of the dark-germinating seeds of 3 varieties of tomato is controlled by the phytochrome system. Germination is inhibited by far red radiation and repromoted by red applied after far red. At low temperatures, 17 to 20°, a single, low energy far red irradiation is sufficient to inhibit germination in all 3 varieties. At higher temperatures far red is less effective in the inhibition of the germination of the tomato seeds. The phytochrome fraction present as P_FR in the dark-germinating seeds of the Ace variety is about 40% of the total phytochrome present.     

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

Control processes in the induction and relief of thermoinhibition of lettuce seed germination : actions of phytochrome and endogenous ethylene

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20°C but was inhibited at 27°C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28°C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32°C. However, the promotive effect of light could be extended to 34°C by repeated irradiations. At any one temperature, increased frequency of irradiations increased germination percentage, and with each degree increase in temperature, increasingly frequent irradiations were necessary to elicit maximal germination. Loss of the effectiveness of single irradiations with increase in temperature may result either from acceleration of the thermal reversion of the far red-absorbing form of phytochrome or decrease in seed sensitivity toward a given percentage of the far red-absorbing form of phytochrome. Using continuous red light to induce germination, the role of endogenous C₂H₄ in germination at 32°C was studied. Ethylene evolution from irradiated seeds began to increase 2 hours prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of C₂H₄ throughout the 24 hour incubation period. Inhibition of C₂H₄ synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C₂H₄ action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C₂H₄ overcame these blockages. The results showed that participation by endogenous C₂H₄ was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C₂H₄ since exogenous C₂H₄ alone in darkness did not promote germination.     

Thermal thresholds as predictors of seed dormancy release and germination timing: altitude-related risks from climate warming for the wild grapevine Vitis vinifera subsp. sylvestris

Background and Aims

The importance of thermal thresholds for predicting seed dormancy release and germination timing under the present climate conditions and simulated climate change scenarios was investigated. In particular, Vitis vinifera subsp. sylvestris was investigated in four Sardinian populations over the full altitudinal range of the species (from approx. 100 to 800 m a.s.l).

Methods

Dried and fresh seeds from each population were incubated in the light at a range of temperatures (10–25 and 25/10 °C), without any pre-treatment and after a warm (3 months at 25 °C) or a cold (3 months at 5 °C) stratification. A thermal time approach was then applied to the germination results for dried seeds and the seed responses were modelled according to the present climate conditions and two simulated scenarios of the Intergovernmental Panel on Climate Change (IPCC): B1 (+1·8 °C) and A2 (+3·4 °C).

Key Results

Cold stratification released physiological dormancy, while very few seeds germinated without treatments or after warm stratification. Fresh, cold-stratified seeds germinated significantly better (>80 %) at temperatures ≥20 °C than at lower temperatures. A base temperature for germination (T_b) of 9·0–11·3 °C and a thermal time requirement for 50 % of germination (θ₅₀) ranging from 33·6 °Cd to 68·6 °Cd were identified for non-dormant cold-stratified seeds, depending on the populations. This complex combination of thermal requirements for dormancy release and germination allowed prediction of field emergence from March to May under the present climatic conditions for the investigated populations.

Conclusions

The thermal thresholds for seed germination identified in this study (T_b and θ₅₀) explained the differences in seed germination detected among populations. Under the two simulated IPCC scenarios, an altitude-related risk from climate warming is identified, with lowland populations being more threatened due to a compromised seed dormancy release and a narrowed seed germination window.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

EFFECT OF HIGH PRESSURE ON GERMINATION OF SEEDS (MEDICAGO SATIVA AND MELILOTUS ALBA)

An increase in percentage germination is obtained with seeds of Medicago sativa exposed for 1 to 10 minutes at 2000 atmospheres hydraulic pressure at 20°C., dried, and germinated after 30 days; and from seeds of Melilotus alba under the same conditions of pressure, when exposed for 5 to 30 minutes, dried, and germinated 30 days later. Exposures to 500 atmospheres pressure was less advantageous for germination; the vitality of seeds normally germinating was more rapidly destroyed than the hard impermeable seeds rendered permeable by the pressure treatment. At 0°C., it required approximately 2½ times the exposure to 2000 atmospheres for seeds of Medicago sativa, and approximately 5 times the exposure for seeds of Melilotus alba, as it did at 20°C.     

Photocontrol of germination in Amaranthus caudatus

Summary Germination of Amaranthus caudatus is inhibited by light, far-red being the most effective part of the spectrum. At temperatures of 25° and below there is a low final germination percentage under continuous far-red whereas above 25° there is only a delaying effect. In the presence of a saturating concentration of gibberellic acid (GA₃) at 25° seeds germinate under continuous far-red although they are delayed. At 25° seeds exposed to 48 hr far-red fail to germinate when transferred to darkness. This induced dormancy can be broken by a single short exposure to red light given at any time after the far-red illumination. This effect of short red can be reversed by a subsequent short period of far-red indicating that the seeds are phytochrome controlled. Although most seeds have escaped from the reversing effect of short far-red after an intervening dark period of 5 hours, germination is greatly reduced by continuous far-red at this time. Results of exposing seeds to varying periods of far-red before and after dark imbibition are interpreted in terms of a continual production of phytochrome in its active P _fr form and a requirement for P _fr action over a long period of time. Effects of intermittent and continuous low intensity far-red on the inhibition of germination provides further evidence for a low energy photoreaction involving phytochrome. Effects on Germination Index of continuous illumination with various light sources maintaining different P _fr /P _total ratios have been investigated. The results suggest that the proportion of phytochrome in the P _fr form is the most important factor in the regulation of germination. A scheme for the phytochrome control of germination in Amaranthus caudatus is presented and possible explanations for the dependence on P _fr /P _total ratio are discussed.Holder of a Science Research Council Studentship.     

Enhanced Phytochrome Sensitivity and Its Reversal in Amaranthus albus Seeds

Seed of Amaranthus alus L. develop an enhanced sensitivity to the farred absorbing form of phytochrome after prolonged imbibition at temperatures >32°C. The enhanced sensitivity developed at 40°C could be reversed by subsequent treatment at 20°C and similarly reestablished by repeating a 40°C treatment. It is concluded that relative sensitivity to the far-red absorbing form of phytochrome may be readily manipulated in seeds of A. albus.     

Seed Germination Ecology of Feather Lovegrass [Eragrostis tenella (L.) Beauv. Ex Roemer & J.A. Schultes]

Feather lovegrass [Eragrostis tenella (L.) Beauv. Ex Roemer & J.A. Schultes] is a C₄ grass weed that has the ability to grow in both lowland and upland conditions. Experiments were conducted in the laboratory and screenhouse to evaluate the effect of environmental factors on germination, emergence, and growth of this weed species. Germination in the light/dark regime was higher at alternating day/night temperatures of 30/20 °C (98%) than at 35/25 °C (83%) or 25/15 °C (62%). Germination was completely inhibited by darkness. The osmotic potential and sodium chloride concentrations required for 50% inhibition of maximum germination were -0.7 MPa and 76 mM, respectively. The highest seedling emergence (69%) was observed from the seeds sown on the soil surface and no seedlings emerged from seeds buried at depths of 0.5 cm or more. The use of residue as mulches significantly reduced the emergence and biomass of feather lovegrass seedlings. A residue amount of 0.5 t ha^-1 was needed to suppress 50% of the maximum seedlings. Because germination was strongly stimulated by light and seedling emergence was the highest for the seeds sown on the soil surface, feather lovegrass is likely to become a problematic weed in zero-till systems. The knowledge gained from this study could help in developing effective and sustainable weed management strategies.     

Studies of the Mechanism of Enhancement of Phytochrome-dependent Lettuce Seed Germination by Prechilling

Temperature and kinetic studies were performed to examine the mechanism by which prechilling stimulates phytochrome-dependent seed germination in lettuce, Lactuca sativa, L. cv. Grand Rapids. Imbibed seeds were given a short far red irradiation and one day of dark incubation at 20 C to establish very low levels of the far red-absorbing form of phytochrome—(Pfr). Germination was greatly stimulated by subsequent prechilling treatments when they were followed by a second short far red irradiation. Prechilling therefore increased germination sensitivity to the low, normally inhibitory Pfr levels established by far red irradiation. This sensitivity increased with lowered prechilling temperature to a maximum near 4 C. It was linearly dependent upon duration of prechilling at 4 C up to a near maximal response at 10 hours, and it decayed in a converse manner when seeds were returned to 20 C after 10 hours at 4 C. Prechilling also increased germination responses to subsequent periods of high levels of Pfr which were initiated by red and terminated by far red irradiations. High Pfr periods adequate to promote the germination of unchilled seeds produced sharp inflections at 18 C in the dependence of germination on prechilling temperature. Rates of phytochrome potentiation of germination were not affected by prechilling. The response to prechilling fit a mechanism involving homeoviscous adaptation of membrane lipids to temperature.     

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

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