Changes in Phytochrome Expressed by Germination of Amaranthus retroflexus L. Seeds

Abstract

Effects of red (600 to 680 nanometers) and far red (700 to 760 nanometers) irradiances on Arnaranthus retroflexus L. seeds indicate that synthesis of phytochrome in the red-absorbing form takes place in water-imbibed nongerminat-ing seeds at 35 C. After 96 hours in darkness, conversion of about 0.10% phytochrome to the far red-absorbing form induces 50% germination. Continuous far red radiation at 35 C with an irradiance of 0.4 X 10-10 Einsteins per square centimeter per second caused photoinactivation of phytochrome about equal to the rate of synthesis. Germination of seeds at 35 C, following far red irradiation adequate to establish the photostationary state, is enhanced by holding at 26 C for 16 minutes. Germination is unaffected relative to controls at constant temperature, if the period at 26 C precedes irradiation. The results indicate a quick response to action of phytochrome in a germination process. In the germination of Amaranthus retroflexus L. seeds, we find several features of control, some of which have been studied in other seeds (6, 10, 12, 13), which might be expected to be general ones. These features include the rates of synthesis, transformation, and destruction of phytochrome and the rate of germination. All of these depend on the temperature, irradiation, times of imbibi-tion, and other conditions. Conversely, the several rates can be estimated from corresponding degrees of germination. Results obtained also bear on a phytochrome control of a response under long irradiation, the so-called high energy reaction (2, 6). MATERIALS AND METHODS Mature Amaranthus retroflexus L. seeds (lot 46) were collected near Beltsville, Maryland, in August, 1966, and held at-10 C in sealed containers. They were germinated in lots of 100 in Petri dishes on filter paper moistened to a shiny appearance with tap water. Seeds were held in darkness in light-tight bags when not being irradiated. Germinated seeds were counted after holding for 2 days in darkness at 35 C upon completion of treatment. Two lots were used for each condition in all experiments. Results presented came from some 200 tests. Red light was either limited spectrographically to the 640 to 680 nm region (Tables I and H) or to the 590 to 680 nm region (Fig. 2, Tables HI and VI) by red cellophane filters with a broad fluorescent source. Far red light in the >700 nm region was obtained with red + blue cellophane filters and incandescent filament sources (Fig. 2, Tables II, HI, and IV) and in the 740 to 800 nm region with a Corning 7-69 (5-mm) glass filter + 30 cm H20 and a carbon arc source (Tables IV, V, and VI). Incident intensities were measured on a wedge interference filter spectroradiom-eter (K. H. Norris) calibrated against a standard lamp. They are expressed in Einsteins cm-2 sec-1 in the defined spectral region (1.0 millijoule = 0.552 X 10 Einsteins sec'l at 660 nm). Light absorption by the dark purplish red anthocyanin of the A. retroflexus seed coat prevents a spectroscopic measurement of phytochrome and decreases the fraction of incident light reaching the site of action. Transmissions of separated seed coats (Fig. 1) were kindly measured by K. H. Norris with a micro-spectrometer. MEASUREMENTS AND PRELIMINARY DEDUCIIONS Unless prechilled (14), only a few of the seeds germinate in darkness. Germination, which requires temperatures >30 C, is enhanced by short exposure to light (9). Pfr, the far red-absorbing form of phytochrome, P, is shown by the prechilhing display to disappear rapidly at 35 C either by reversion to the red-absorbing form, Pr, or by destruction of P (14). Full germination can be reached with 1.2 X 101 Einsteins cm-2 red irradiation (590-680 nm steady state) after 36 hr of dark imbibition at 35 C. Unlike other seed kinds, seeds of A. retroflexus do not decline in responsiveness to red radiation after prolonged imbibition (9). On the contrary, the responsiveness of the seeds to a given irradiance of red increases as dark imbibition at 35 C continues from 24 to 96 hr. Thereafter, the response is constant to 140 hr (Table I). About 4-fold greater red irradiance is required at the 48th hr than at the 96th hr to induce a given germination. A striking feature of the results is that germination is greatly enhanced after 96 hr by transformation of a calculated <0.1 % Pr to Pfr even if seed coat absorption of light is ignored. The major interpretation is that synthesis of Pr continues with time of imbibition in darkness even though the seeds remain dormant. The [Pfr] level (brackets indicate concentration), resulting from a given red irradiation, accordingly increases. Seeds imbibed for 96 hr in farred have a lower germination percentage after a red irradiance than those held in darkness (Table II). The change in sensitivity is intensity-dependent. The interpretation is that destruction of P by light depends upon the rate of excitation of P. Results similar to those in Tables I and H are shown from other experiments in Figure 2 and Table II. The results in Table Ill also show continued change of [P] under further periods of darkness or far red irradiation after initial treatments for 48 or 96 hr. These several experiments show that [P] after 96 hr darkness at 35 C is such that transformation of Pfr by far red irradiation adequate for a photosteady state (1-2% Pfr) should be adequate to enhance germination greatly.