Abstract— The exact quantity of phytochrome in crude homogenates (2kS) prepared from embryonic axes of Pisum sativum during imbibition at 25°C on 0,2% agar was estimated optically. The problem of the scattering factor was solved by using highly purified phytochrome as an internal standard. The content of phytochrome protein moiety in diluted samples of the crude homogenates of the axes was also determined by an enzyme‐linked immunosorbent assay (ELISA). Phytochrome was not detectable either spectropho‐tometrically or immunochemically in 2kS of dormant dry axes. Embryonic axes quickly absorbed water during the first1–2 h after the start of imbibition, after which the fresh weight stayed at a constant level for a further 10 h. The content of spectrophotometrically detectable phytochrome increased during imbibition in the dark, reaching about 0.2 μ.g/axis after 12 h. The amount of phytochrome in 2kS of axes in the light was so small that only about 0.05 μg/axis was detected after 12 h. The content of immunochemically detectable phytochrome greatly increased up to ca. 0.5 μg/axis after 12 h of dark incubation. In 2kS of the light‐grown axes the content of the phytochrome protein was ca. one fourth lower than in dark‐grown axes. We conclude that the appearance and increase of phytochrome in fragments of imbibed embryonic axes were caused by de novo synthesis and that the contents of both photometrically detectable phytochrome and its protein moiety in the light‐grown samples were lower than those in the dark throughout the early germination process.

Soyasaponin 1, a triterpenoid saponin isolated from etiolated pea (Pisum sativum cv. Alaska) shoots and identified as Pfr killer, was examined for its effects on spectral properties of undegraded pea phytochrome. When soyasaponin I in concentrations of 100 micromolar or lower was added to Pr in the dark, the spectrum of Pr was not significantly affected, whereas in the presence of 120 micromolar or higher concentrations the absorption maximum of Pr shifted from 666 to 658 nanometer with slight decrease of absorbance. After a brief exposure of the mixture to red light, the increase in absorbance at 666 nanometers that occurs in the dark was inhibited at 26 micromolar and higher soyasaponin I concentrations; the maximum effect being reached at about 180 micromolar. The decrease in absorbance at 724 nanometers in the dark after red light irradiation was somewhat inhibited by 60 micromolar and totally prevented by 410 micromolar soyasaponin 1. When P658 was irradiated with red light in the presence of 220 micromolar or higher soyasaponin I concentrations, a bleached form (Pbh) was produced instead of Pfr. Phi showed no dark spectral changes, and the phototransformation of Phi to P658 required a significantly high irradiance of far-red light. When the saponin was added to Pfr in the dark, none of the above-described spectral changes occurred, although the same effects were observed after the mixture was exposed briefly to far-red light followed by red light.In the early days of phytochrome study, Furuya and Hillman (6) found that crude aqueous extracts of etiolated pea tissue contain a substance which almost instantaneously destroys phytochrome photoreversibility in the Pfr but not in the Pr form. The existence of such a substance, 'Pfr killer,' was confirmed in the extract of pea but not in A vena by Fox (2). Although Shimazaki and Furuya (11) Hillman, who played a critical role in the study of photomorphogenesis and also has discovered the Pfr killer effect with M.F. at Brookhaven two decades ago.

Treatment of imbibed embryonic axes taken from seeds of Pisum sativum with N‐phenylimide S‐23142, a herbicide that has been suggested to inhibit protoporphyrin synthesis, or with N‐methyl mesoporphyrin IX, an inhibitor of the iron chelatase for heme, resulted in a significant decrease in the amount of spectrophotometrically detectable phytochromc in the axes in both cases. However, the amount of immunochemically detectable phytochrome was not affected by either treatment. If S‐23142 inhibits the synthesis of protoporphyrin IX in pea, it appears that the conversion of protoporphyrinogen IX to protoporphyrin IX is involved in the biosynthesis of the phytochrome chromophore. The conversion of protoporphyrin IX to heme (Fe‐protoporpbyrin) also appears to be a step in the biosynthesis of the chromophore, since N‐methyl mesoporphyrin IX prevented the synthesis of spectrophotometrically detectable phytochrome but did not affect the magnesium chelatase activity required for the synthesis of chlorophyll in pea embryonic axes. The results suggest that protoporphyrinogen IX, protoporphyrin IX and heme are intermediates in the biogenesis of the phytochromc chromophore. The pathway to phytochromobilin might become fixed after protoporphyrin IX, being directed toward the Fe branch for heme rather than to the Mg branch for chlorophyll.