Far-red Absorbing Form Of Phytochrome Research Articles

Environmental conditions and endogenous mechanisms involved in secondary dormancy of seeds

ABSTRACT Secondary dormancy, is defined as the dormancy developing in seeds after harvest or dispersal. Under field conditions secondary dormancy is part of cyclic changes in the level of dormancy that often follow a seasonal pattern. It develops when germination is inhibited, e.g. by burial in soil. Temperature, absence of light or oxygen, presence of volatile or allelopathic inhibitors and moisture conditions are among the factors which may contribute to inhibition of germination and thus to development of dormancy. Decreasing light sensitivity is part of the development of secondary dormancy. It is partly attributed to photo- or dark-reversion of the far-red absorbing form of phytochrome (Pfr), pre-existing in the seeds or newly formed, to Pr. Changing levels of Pfr at its primary site of action, possibly being a membrane, are also held responsible for the decreasing light-sensitivity. Secondary dormancy is not related to a deterioration of the seeds. A specific relationship with any particular metabol...

Control Processes in the Induction and Relief of Thermoinhibition of Lettuce Seed Germination

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20 degrees C but was inhibited at 27 degrees C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28 degrees C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32 degrees C. However, the promotive effect of light could be extended to 34 degrees 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 C(2)H(4) in germination at 32 degrees 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 C(2)H(4) throughout the 24 hour incubation period. Inhibition of C(2)H(4) synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C(2)H(4) action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C(2)H(4) overcame these blockages. The results showed that participation by endogenous C(2)H(4) was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C(2)H(4) since exogenous C(2)H(4) alone in darkness did not promote germination.

Phytochrome ? all regions marked by a set of monoclonal antibodies reflect conformational changes

Monoclonal antibodies to defined locations on six regions of the phytochrome molecule (from Avena sativa L. or Zea mays L.) were each found to have a different affinity toward the farred-absorbing form of phytochrome (Pfr) and the red-absorbing form (Pr). The differences were small, but were consistently shown by antibodies which bind to the vicinity of the aminoterminus, the carboxylterminus and to sequences in between. It seems that the conformational differences between Pr and Pfr extend over the whole molecule in as far as it is represented by these regions and the antibodies binding to them.

A Kinetic Analysis of Phytochrome Controlled Mesocotyl Growth in Zea mays Seedlings

Mesocotyl elongation in 4 day old etiolated seedlings immediately following 3 hours of white light (3 h W) is reversibly controlled by phytochrome. Time-lapse video measurements were made of the 5 millimeter zone just below the coleoptile which is the main growth region of the mesocotyl. The growth kinetics were determined for five contiguous 1 millimeter zones subtending the coleoptile node for nonirradiated seedlings, for seedlings given 3 h W, and 3 h W followed by terminal far-red (FR) or red subsequent to the far-red (FR/R) irradiation. Each zone in nonirradiated seedlings exhibits exponential elongation kinetics during the early stages of elongation. This finding suggests that during elongation, a growth limiting factor is also exponentially increasing. Following 3 h W differences in the kinetic responses were found for each zone. In all zones, the inhibitory effect following the 3 h W is totally FR reversible. The effect of FR is reversed by R. The upper zone exhibits the fastest response and is the most plastic in its growth response. The three upper zones all exhibit spontaneous and sharp recoveries with time. It is suggested that the control by phytochrome is not inductive but rather continuous, the controlling factor being either the level of the far red-absorbing form of phytochrome (Pfr) or the ratio Pfr to total phytochrome.

Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate, phytochrome and photooxidative damage of plastids

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that 'intact plastids' are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some 'plastidic signal' is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state (ϕ) is not linear; all values of ϕ from 0.004 to 0.13 promote growth maximally, in the range of values of ϕ from 0.13 to 0.22 there is a linear growth response, between values of ϕ of 0.22 and 0.35 there is again no differential effect, and for ϕ values above 0.35 there is a strong (near linear) effect of ϕ on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day ϕ is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day ϕ, namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.

Photocontrol of hypocotyl elongation in light-grown Cucumis sativus L.

The control by phytochrome of hypocotyl elongation of light-grown Cucumis sativus L. after a white-light period was examined. The farred-absorbing form of phytochrome inhibits hypocotyl elongation. The response to phytochrome photostationary state (ϕ) is not linear; all values of ϕ from 0.004 to 0.13 promote growth maximally, in the range of values of ϕ from 0.13 to 0.22 there is a linear growth response, between values of ϕ of 0.22 and 0.35 there is again no differential effect, and for ϕ values above 0.35 there is a strong (near linear) effect of ϕ on elongation. A kinetic examination of events following the white-light period shows that the major recovery from the photoperiod requires 8.5 h of darkness. End-of-day far-red treatment produces a very different response pattern, with a minor growth stimulation within 28 min of treatment followed by a major effect after 80 to 90 min. Three hours after far-red treatment there is a transient decline in growth rate which persists for about 2 h. Over the whole time course there is a great stimulation of growth rate compared with the controls. A similar growth-rate pattern also occurs if the end-of-day ϕ is 0.48, although the magnitude of the growth stimulation is less. Two components are affected by end-of-day ϕ, namely the time at which growth recovers and the subsequent growth rate. In the long term, the latter accounts for most of the differences in elongation growth. The dark recovery when only the hypocotyl is irradiated requires 4 h, but end-of-day far-red treatment reduces this to about 1.5 h. The persistence of the far-red-absorbing form of phytochrome for many hours in darkness in these light-grown plants is also demonstrated.

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.

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.

Acceleration of Dark Reversion of Phytochrome in Vitro by Calcium and Magnesium

Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples.

Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.

Loss of Phytochrome Photoreversibility in Vitro

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, "Killer," which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.

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.

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.