Richard Yu

AN ACTION SPECTRUM OF PHYTOCHROME BINDING TO A SUBCELLULAR FRACTION OF MAIZE COLEOPTILES

Abstract— An action spectrum was constructed for the photo‐induced pelletability of phytochrome in excised coleoptiles of etiolated maize seedlings. It closely resembled the absorption spectrum of purified phytochrome in the P, form as reported in the literature. The spectral dependence of phytochrome pelletability effected by sustained irradiation (4 h) was also determined and it appeared remarkably similar to the high irradiance response (HIR) action spectrum reported for the inhibition of lettuce hypocotyl lengthening. The induction action spectrum was held to support the conclusion that phytochrome itself is the photoreceptor for its own binding to the subcellular fraction of maize coleoptiles and that the binding phenomenon is an early, if not the first, physiological consequence of irradiation. Also a modified version of Hartmanns interpretation of the high irradiance response was given.

Far red and blue light-induced binding of phytochrome to a subcellular fraction of maize coleoptiles

Summary Irradiation of excised maize coleoptiles with 660 nm light caused phytochrome to become associated with a crude membrane fraction when the tissues were subsequently fractioned in the presence of added Mg++ ion. Over 85% of the particle-associated phytochrome was found to be in the far red absorbing form (Pfr). Wavelengths in the blue and far red ranges were found to be effective in inducing high levels of particle-associated phytochrome, though less efficiently than red light. Relatively longer irradiations with blue or far red light were therefore required to effect an equivalent degree of phytochrome-particle association when compared with 660 nm light. Examination of the phytochrome state in the crude membrane fraction of coleoptiles irradiated with large doses of far red light showed no detectable amount of Pfr. In the case of coleoptiles irradiated with blue light, about 40–50 % of the particle bound phytochrome was in the Pfr form. The data are interpreted as supporting a working hypothesis that any phytochrome molecule (irrespective whether Pfr or Pr) can be activated to associate with its putative binding site by absorbing sufficient energy and that this can be considered a primary photoreaction.

Phytochrome Binding Induced by Irradiation of Extract of Maize Coleoptiles

Summary Phytochrome binding was induced by irradiating extracts of maize coleoptiles but at a much lower efficiency when compared with that obtained by irradiating the intact tissues. Percentage of total phytochrome bound to a particulate fraction by in vitro or in vivo irradiation was only slightly affected by the ratio of fresh tissue weight : extraction buffer volume used in homogenization and fractionation of the tissue. The bound phytochrome once induced by in vivo irradiation is stable for a brief period and resistant to displacement by exogenous phytochrome. Phytochrome binding can be effected by in vitro or in vivo irradiation at 0 °C. When phytochrome binding is induced in vitro at temperatures above 0 °C changes occur which apparently do not take place when binding is similarly induced by in vivo irradiation. In vivo and in vitro induction of phytochrome binding differs also in that in vivo -induced binding is inhibited by azide or cyanide whereas in vitro -induced binding is not.

Effect of cyanide and azide on phytochrome-binding site interaction

Summary Cyanide and azide have been used to study the causal relationship between phytochrome-binding site interaction and phytochrome destruction in maize coleoptiles. Although these inhibitors did not appear to affect the initial binding or attachment of phytochrome to its binding site in red irradiated coleoptiles they nevertheless curtailed the dynamic interaction between phytochrome and binding site subsequent to their initial union. This interaction, presumably a temperature dependent process, did not take place at 0°C, hence cyanide or azide was without effect at that temperature. Besides reducing phytochrome pelletability, cyanide or azide also affected cellular respiratory activity; inhibition of both reactions occurred rapidly and it is envisaged that cellular respiration is involved in phytochromebinding site interaction.

The active form of phytochrome: a new hypothesis based on phytochrome pelletability studies.

Difficulties arising from the current dogma that the far-red absorbing form of phytochrome (Pfr) is the only active form are discussed. A new hypothesis is proposed in which phytochrome is held to be the photoreceptor for both low energy (pulse) and high energy (HIR) responses. There is a common basic mechanism of action involving interaction between phytochrome and a binding site within the cell. The phytochrome involvement in low energy responses exhibits an action spectrum for binding that matches the Pr absorption spectrum and reversibility by far-red irradiation. Upon prolonged irradiation the phytochrome-binding site interaction acquires different characteristics that are reminiscent of those displayed in HIR, e.g. dependence on sustained irradiation for continual binding, dependence of the degree of binding on irradiance and the similarity of the action spectrum with that of HIR action spectra, e.g. that for inhibition of lettuce hypocotyl lengthening. As expected on the basis of the new hypothesis the particulate fraction of phytochrome contains both Pr and Pfr. Arguments are advanced that the presence of Pr in pellets of particulate phytochrome cannot be accounted for by (i) the “induced fit” hypothesis, (ii) the “pigment cycling” hypothesis, and (iii) the “open phytochrome-receptor model”. We conclude that phytochrome molecules, after being sufficiently energized can interact with their intracellular binding sites irrespective of their chromophoric configuration.

In vitro Crosslinking of Phytochrome to its Putative Receptor with Bi-imidoesters

Summary Covalent linkage of phytochrome to a subcellular particulate fraction was achieved in vitro by reacting a cell-free homogenate of red light irradiated coleoptiles, extracted in a buffer containing Mg ++ at 0°C with a bi-imidoester. Three homologous bi-imidoesters [dimethyl suberimidate (DMS), dimethyl adipimidate (DMA), and dimethyl 3,3′-dithiobispropionimidate (DTBPI)], were used of which DMS and DTBPI were more effective than DMA. Particulate phytochrome crosslinked by DTBPI was released into solution when the reaction product was treated with dithiothreitol. Successful crosslin king depended on red light irradiation of the tissue and the presence of Mg ++ in the buffers used for homogenizing the tissue and for carrying out the crosslinking reaction.

Characteristics of phytochrome and binding site interaction under high irradiance conditions

Summary Some characteristics of pelletable phytochrome obtained from maize coleoptiles irradiated by different light regimes are compared. The general sedimentation behaviour and buoyant density of the phytochrome-containing particles, and the chromatographic elution behaviour of particulate phytochrome show no apparent difference when prepared from coleoptiles irradiated by pulse red or by prolonged far red or blue light. The similarity in behaviour supports the hypothesis that the interaction between phytochrome and binding site(s) in maize coleoptiles is identical irrespective of the type of irradiation.

Effects of 2,4-Dinitrophenol and Azide on Phytochrome and Binding Site Interaction in Coleoptiles and Primary Leaves of Maize and Hypocotyl Hooks of Zucchini

Summary 2,4-dinitrophenol and azide have been used in studying characteristics of the phenomenon of phytochrome and binding site interaction in maize coleoptiles and primary leaves as well as in zucchini hypocotyl hooks. Changes in response to the inhibitors were observed in initial phytochrome binding before dark incubation, total cellular phytochrome and the maintenance of phytochrome binding during incubation in darkness of red light irradiated tissues. In the three plant tissues studied, when destruction of phytochrome was inhibited by dinitrophenol or azide, these compounds appeared to enhance phytochrome binding during dark incubation. While dinitrophenol or azide did not appear to inhibit initial phytochrome binding in zucchini hypocotyl hooks, it did so strongly in maize coleoptiles and weakly in maize primary leaves.

Phytochrome does not associate with ribonucleoprotein particles from Cucurbita pepo

Summary Following red light irradiation of excised zucchini hypocotyl hooks and tissue homogenization in a buffer containing EDTA about 50% of the cellular phytochrome became sedimentable into a crude mitochondrial fraction. Upon further analysis by sucrose gradient centrifugation, phytochrome in the crude mitochondrial fraction was distributed near the mitochondrial region and the gradient/sample interface of the gradient. Neither of these two areas of phytochrome activity exactly coincided with the RNA-containing material. Treatment of the crude mitochondrial fraction with Triton ×100 solubilized the phytochrome, RNA-containing material, and the NADPH cytochrome c reductase activity but not the cytochrome c oxidase activity. Under this condition phytochrome and RNA-containing material still appeared to sediment separately. When homogenized in the presence of EDTA, phytochrome and RNA-containing material remaining in the post-mitochondrial supernatant were eluted as separate zones from Sepharose 4B/CL columns, phytochrome being the slower eluting material. When red light irradiated hooks were homogenized in a buffer containing Mg ++ , phytochrome in the post-mitochondrial supernatant was found to elute faster than the RNA-containing material. But in neither case were phytochrome and RNA-containing material eluted together. Pretreatment of red lightirradiated hooks with glutaral-dehyde resulted in most of the phytochrome in the crude mitochondrial fraction to sediment apparently with the cytochrome oxidase activity and RNA containing material. Triton ×100 treatment of the crude mitochondrial fraction did not appear to solubilize the phytochrome and RNA-containing material but caused them to sediment separately. They banded with the cytochrome c oxidase activity on prolonged centrifugation. In post-mitochondrial supernatant from glutaraldehyde-treated tissues, phytochrome and RNA-containing material were eluted from Sepharose columns as separate zones, phytochrome being the slower component.

Covalent Crosslinking of Phytochrome and its Binding Site: a Comparative Study with Different Plant Tissues under in vivo and in vitro Conditions

Summary Chemical crosslinking of phytochrome to its binding site(s) was effected using red light irradiated intact tissues (maize coleoptiles and primary leaves, zucchini hypocotyl hooks, barley and oat shoots) or cell-free homogenates derived from them. The following crosslinking reagents were tried: bi-imidoesters (dimethyl adipimidate, dimethyl suberimidate, and dithiobispropionimidate) or glutaraldehyde. Three patterns of responsiveness were observed. Maize coleoptile is responsive to bi-imidoesters and glutaraldehyde under in vivo and in vitro conditions. Zucchini hook is responsive to glutaraldehyde but not to biimidoesters, under in vivo and in vitro conditions. The consistency in the pattern of result obtained under in vivo and in vitro conditions for maize coleoptile and zucchini hook suggests a difference between phytochrome and binding site interaction in these two organs. In general, intact tissues of the cereal leaves are not responsive to bi-imidoesters or glutaraldehyde. On the other hand, substantial enhancement of phytochrome pelletability was observed when cell-free homogenates derived from red light irradiated cereal leaves were treated with crosslinking reagents in vitro . The lack of response of intact cereal leaves to these reagents might be due to cellular impermeability.