We have examined phytochrome effects on the abundance of transcripts from several nuclear and chloroplast genes in buds of dark-grown pea seedlings and primary leaves of dark-grown mung-bean seedlings. Probes for nuclear-coded RNAs were selected from a library of cDNA clones and included those corresponding to the small subunit (SS) of ribulosebisphosphate carboxylase and a chlorophyll a/b binding protein (AB). Transcripts from chloroplast genes for RuBP carboxylase large subunit (LS) and a 32,000-dalton photosystem II polypeptide (PII) were assayed with cloned fragments of the chloroplast genome. In addition, we present data on transcripts from a number of other nuclear genes of unknown function, several of which change in abundance during light-induced development. Transcript levels were measured as a proportion of total RNA by a dot blot assay in which RNA from different tissues or stages is fixed to nitrocellulose and hybridized with (32)P-labeled probes prepared from cloned DNAs. Several patterns of induction can be seen. For example, although both SS and AB RNAs show positive, red/far-red reversible responses in both pea and mung bean, in pea buds the induction ratio for SS RNA is much higher than that for AB RNA, while just the reverse is true for mung-bean leaves. In addition, treatment with lowfluence red light produces full induction of the pea AB RNA, while SS RNA in the same tissue does not reach a maximum steady-state level until after about 24 h of supplementary high-intensity white light. In pea buds, chloroplast genes (LS, PII) also show clear responses to phytochrome, as measured by the steady-state levels of their RNA products. Chloroplast DNA levels (as a fraction of the total cellular DNA) show the same response pattern, which may indicate that in peas many of the light effects we see are related to a general stimulation of chloroplast development. In mung beans, the levels of plastid DNA and RNA are already quite high in the leaves of 7-d dark-grown seedlings, and light effects are much less pronounced. The results are consistent with the notion that chloroplast development is arrested at a later stage in dark-grown mung-bean leaves than in etiolated pea buds.
Abstract. A method has been developed whereby the second positive phototropism can be observed separately from the first positive and negative phototropic responses w-hioh also oocur in oat coleoptiles. Although the second positive phototropic response has often been referred to as the base response, photoreception for it is shown to occur mainly in the apical 3 mm of the coleoptile. The Bunsen-Roscoe reciprocity law, so typical of first positive phototropism, does not apply to the second positive responses, and the amount of curvature increa.ses linearly with the duration of the stimulus. However, although this linear proportionality between stimulus duration and response is the major factor determining response at all intensities tested, the inten,sity of the stimulus does influence the Tesponse somewhat. The action spectrum for the response shows no activity above 510 nm and 'has peaks at 375 and 450 nm. In all but one particular it closely resembles the aotion spectrum ifor the first positive phototropism, and it is conoluded that the same, or similar, pigments may well be the photoreceptors for both types of response. The identity of this blue light absorbing pigment is not known.De-spite the availability of action spectra for the first positive phototropism in oats (15,17). the photoreceptor for this response has not been definitely characterized (9,17). Furthermore, the earlx experiments on phototropism demonstrated that in addition to first positive phototropic curvatures, oat coleoptiles also exhibit negative and second positive types of response (1, 4). From a study of th-e doseresponse curves for phototropism in Avena and of theoretical models for these data, Zimmerman and Briggs (19,20) suggested that Avena coleoptiles possess 3 different photoreceptor systems for the 3 types of phototropic response-first positive, negative, and second positive. If this be the case, then the photoreceptive pigment for the second positive type of curvature would he di'fferent from that for the first positive phototropism and might be identified from an action spectrum for the secolnd positive response. For this reason, and in order to characterize the little-known second positive response more clearly, the present experiments on second positive p)hototropism in coleoptiles of Are(nMa wer-e undertaken.Although the second positive phototropic response, which occurs with large stimulus energies or long stimulus durations, has not beeli extensively studied, there are strong indications that the B3unsen-Roscoe reciprocity law, which holds for first positive phototropism, is not applicable to it (2, 5. 17 ((20) subtracted the responses predicted by their theor-etical models for first positive and negative l)hototropism from their phototropic dose-response curves, the remaining responses, assume(d to be of the second positive type, increased linearly xvith stimulus duration and were not affected by a 10-fol(d vtariation in intensity. Unfortunately the overlap of first positive responces bv second positive otnes when low i...
The transformation difference spectrum for phytochrome (Pr spectrum minus Pfr spectrum) in pea tissue is determined below 560 nanometers and compared with similar data on phytochrome in vitro The difference spectrum in vivo between phytochrome intermediates and Pfr is also shown for comparison with the data on phytochrome solu. tions. These comparisons show that the peaks in the spectra occurring in the blue wave lengths are shifted to shorter wave lengths and are much enhanced when phytochrome is extracted from the cell and placed in solution. The results indicate that the physicochemical state of phytochrome in the cell may be different from that of the extracted pigment. about 0.1 OD per A (A OD) of phytochrome. A typical tissue sample for spectrophotometric observation could contain 0.2 A (A OD); thus the change in optical density to be expected is of the order of 0.02 OD in a region where the total optical density is between 2 and 3.Since we had available a spectrophotometer designed to measure small absorbancy changes in dense tissue samples, we attempted to observe absorbancy changes related both to phytochrome transformation and to the long-lived phytochrome intermediates described by Briggs and Fork (3,4) at wave lengths below 560 nm. Such observations might provide information on the state of phytochrome in vivo and could partially explain the difference in the relative effectiveness of blue light for transformation in vivo and in vitro. MATERUILS AND METHODSIn many physiological responses mediated by phytochrome, it has been shown that blue light can evoke reactions similar to those caused by red and far red light (1, 2, 13). Such observations led to the hypothesis that both forms of phytochrome, Pr and Pfr, absorb blue light (1, 2). With the development of methods for extracting phytochrome, it was indeed found that the pigment has absorption maxima in the blue spectral regions -Pr at about 370 nm and Pfr at about 400 nm (7,11,16 Plant Material. Peas (Piswn sativum L. cv Alaska) were soaked in tap water for 4 hr and planted on moist cellulose packing material, Kimpak 6223 (Kimberly-Clark), in plastic refrigerator boxes. The plants were grown in complete darkness at 25 C and 80% relative humidity for 3 days, although occasionally 2-or 4-day-old plants were used. To prepare a sample, the apical hook regions (5 mm) were harvested under dim green light, the plumules were removed, and the sections were placed in a beaker on ice. Approximately 150 such sections were chopped into pieces about 1 mm3 and placed in a chilled cuvette 1.6 cm in diameter. The sections were gently packed so that light attenuation through the sample was uniform. Total phytochrome content uncorrected for incomplete photoconversion by red light was measured from absorption spectra of the samples after saturating red and far red irradiation. The average photoreversible phytochrome content of such samples varied between 0.15 and 0.20 A (A OD).Phytochrome Isolation. Phytochrome from peas was obtained by minor modification of met...
Radish seedlings (Raphanus sativus L.) were grown for 4 days in complete darkness, or in white light, or for 3 days in darkness followed by 1 day of red light. Phototropic dose-response curves for the seedlings grown in these three ways were determined with 460-nm light. Dicot seedlings have occasionally been used for studies of phototropism. Most of these experiments relate to the response mechanism; auxin and gibberellins have been investigated as the causal agents of the unequal growth which leads to phototropic bending (2,5,6,8, 10). However, little information was available on the characteristics of the photoreceptor in these seedlings until Steyer (9) published dose-response curves for phototropism for 13 kinds of dicot seedlings. In these experiments, all the dark-grown seedlings were found to have first positive, then indifferent, and then second positive types of response as the energy in the light stimulus was increased. Steyer's studies indicate that, in sensitivity and speed of response, dicot seedlings are comparable to coleoptiles and that these seedlings would be good objects for more detailed experiments. The Fenway, Boston, Mass. 02115. This paper reports on dose-response curves for phototropism in radish seedlings grown under three light regimes. MATERIALS AND METHODSRadish (Raphanus sativus L.) seeds, Burpee's white radish, were soaked for 2 hr in distilled water, placed in a 5% solution of commercial bleach for 5 min, and then rinsed in distilled water. The seeds were spread out in shallow enamel pans on Kimpak-brand absorbent paper moistened with distilled water. The pans were covered tightly with aluminum foil and placed in a dark cabinet in a room maintained at 25 C. Approximately 24 hr after soaking, seedlings with radicals 3 to 5 mm long and with seed coats removed were selected for transfer to individual metal vials. The vials were 2.8 cm deep and 1.8 cm in diameter and fit snugly into metal racks. Each vial had a handle so that, when placed in the rack, it could be rotated 900 by means of the handle and guides on the rack. The vials were lined with aluminum foil and filled with moist vermiculite.The 24-hr-old seedlings were either grown in the dark, or grown in the dark and then red light treated, or grown in white light. For dark growth, seedlings were transferred to vials under a photomorphogenically inactive dim green safelight described by Withrow and Price (11). The racks holding these seedlings were then placed in deep enamel pans; the pans were covered with aluminum foil and placed in a dark cabinet at 25 C. Seedlings to be treated with red light were transferred to vials under a green light described by Shen-Miller and Gordon (7). The racks of vials were then placed in an enamel pan covered with aluminum foil in a dark cabinet at 25 C. Approximately 72 hr after soaking, the aluminum foil was removed and clear plastic wrap was placed over the pan. The seedlings were then exposed to red light at an intensity of about 0.6 mw cm-2 as described by . After 24 hr of red light,...
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