Plexiglas (Rohm and Haas No. 2423). Light intensities were measured before each experiment with an Eppley eight-junction bismuth-silver thermopile and ...
Plant Physiol. (1980) 66, 1024-1026 0032-0889/80/66/ 1024/03/$00.50/0
Phytochrome Responses to End-of-Day Irradiations in Lightgrown Corn Grown in the Presence and Absence of Sandoz 9789' Received for publication February 6, 1980 and in revised form May 30, 1980
HOLLY L. GORTON AND WINSLOW R. BRIGGS Carnegie Institution of Washington, Stanford, California 94305 ABSTRACT Corn seedlings were grown in white light in the absence and presence of the chlorosis-inducing herbicide San 9789. The resulting green and achlorophyllous seedlings were used to investigate phytochrome-mediated responses to end-of-day far red irradiation and reversal of these responses by subsequent red irradiation. Mesocotyl and coleoptile elongation increased in response to end-of-day far red irradiation, whereas the anthocyanin content of the coleoptiles was decreased. All three responses were reversible by red irradiation following the far red. Dose-response curves for far red induction and red reversal of these responses did not differ significantly for plants grown in the presence or absence of San 9789. Thus, San 9789 appears to affect neither phytochrome itself nor the response system involved. Chlorophyll screening likewise does not affect phytochrome relationships for these responses.
responses should also be unchanged. The dose-response relationships of several responses of corn seedlings, grown with and without San 9789, to end-of-day FR and R light treatments have been examined.
MATERIALS AND METHODS Corn seeds (Zea mays, Bear hybrid WF9X38) were imbibed for 3 h either in H20 or in a suspension of San 9789 in H20. They then were sown on Kimpak moistened with H20 or the appropriate San 9789 suspension and placed in the dark to germinate. After 21 h in the dark, the seedlings were placed on an 8-h light/ 16-h dark regime for the remainder of the growth period. Five 40w Sylvania cool-white fluorescent lamps illuminated the plants during the daily 8-h light periods with an intensity of 19 w m-2 During germination and subsequent growth, the temperature was maintained at 27 C. Forty-eight h after the start of imbibition, the seedlings were transferred from the Kimpak to vials containing 0.65% agar with or without San 9789. Selection for uniform growth occurred at this stage; seedlings with roots about 2 cm long were chosen for transfer. After transfer to agar, seedlings were handled One of the most useful techniques in unravelling problems of in batches of 18 for the duration of the experiment. phytochrome physiology in etiolated seedlings has been the comFR and R treatments as indicated below were given at the end mon dual-wave-length spectral assay for phytochrome. Until re- of the second, third, and fourth 8-h white-light periods. FR was cently, direct measurements of phytochrome in vivo in light-grown provided by 2 GTE Sylvania high-output fluorescent bulbs (F48 seedlings have been impossible because Chl interferes with the T 12/E-5346/VHO) with maximum emission at 745 nm. One layer spectral assay. Screening by Chl prevents complete photoconver- of 3-mm thick "black" Plexiglas (FRF 700, Westlake Plastics, sion of phytochrome and Chl fluorescence causes an apparent Lenni Mills, Pa.) blocked radiation with wavelengths shorter than decrease in the signal size (10, I 1). Jabben and Deitzer (6, 7) have 700 nm. When R reversibility of the effects of end-of-day FR was developed a method to extend the spectral assay to light-grown tested, R was provided by 4 Sylvania warm-white fluorescent plants through the use of the chlorosis-inducing herbicide San bulbs (F20T 12WW). The red filter was a single layer of red 9789. The herbicide blocks carotenoid synthesis (1, 2) so that Chl Plexiglas (Rohm and Haas No. 2423). Light intensities were is not protected from photooxidation. At high light intensities, measured before each experiment with an Eppley eight-junction therefore, plants which have been treated with San 9789 frequently bismuth-silver thermopile and Keithley microvoltmeter (150A). contain negligible amounts of Chl (7). For the lower intensities, a multiplier photometer model 520-M San 9789 shows great promise as a tool for studying phyto- (Photovolt Corporation, New York) calibrated against the therchrome in light-grown plants, but as yet there is no firm evidence mopile, was used. For both instruments, linearity of response was that the over-all sensitivity of the photoreceptor and response confirmed with neutral density filters. systems is unaltered by the herbicide itself. Jabben and Deitzer Dose-response curves for the effect of end-of-day FR irradiation (8) have examined several phytochrome-mediated photomorpho- on mesocotyl and coleoptile length and anthocyanin accumulation genic responses in various seedlings grown with and without San in the coleoptile were obtained by holding the fluence rate of the 9789 in the dark and in the light. They saw no difference in the light constant and varying the time of exposure. Dose-response responsiveness of treated and untreated plants. However, only a curves for R reversal of these effects were similarly established. single light dose, given at a single intensity, was used to elicit each Irradiations were from the side. response. It is possible that the sensitivity of the phytochrome Seedlings were harvested after 5 days of growth, 16 h after the system could be altered by the herbicide, even though, at one light third end-of-day light treatment. Seedlings of this age were found dose, treated and untreated seedlings respond identically. If the to show optimal responses. These responses were cumulative, so photoreception and response systems are unchanged by the her- 3- or 4-day-old seedlings which had received only one or two endbicide, then the dose-response curves for phytochrome-mediated of-day light treatments showed less total response than 5-day-old seedlings. Older seedlings were not used because elongation of the 'Carnegie Institution of Washington publication 695. mesocotyl and coleoptile ceased after 5 days. Mesocotyls and 2 Abbreviations: San 9789, 4-chloro-5(methylamino)-2-(a, a, a-trifluoro- coleoptiles were dissected from the seedlings and photocopied on a Xerox copier to facilitate length measurement (to at least the m-tolyl)-3(2H)-pyridizinone; R, red light; FR far red light. 1024
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Physiol. Vol. 66,
1980
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FIG. 1. Dose-response curves for responses to end-of-day FR. In each case, data for plants grown with ( ) and without (-- -) San 9789 are shown. Controls which received no FR are represented by horizontal lines (D). Irradiation times at 3.2 nE cm-2 s-' ranged from 2 s to 35 min. The responses examined are the decrease in the anthocyanin content of the coleoptile (A) and the increase in mesocotyl (B) and coleoptile (C) lengths. Error bars in B and C indicate SE.
Coleoptiles then were chopped in approximately 2-mm sections and lyophilized. The anthocyanins then were extracted from 50 mg chopped, dried coleoptiles with 3 ml solvent (I -propanol-HCI-H20, 18:1:8 1) for 12 to 24 h at 4 C. Extracts were filtered through a Whatman GF/A glass filter and a Millipore HA filter to minimize turbidity. Absorbance at 522 nm was determined as a measure of the anthocyanin content of the coleoptiles. In vivo absorption spectra of single leaves were obtained with a nearest 0.5 mm).
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FIG. 2. Dose-response curves for R reversal of effects of end-of-day FR (250 nE cm-2 over 38 s). Data for San 9789-treated ( ) and untreated (- -) plants are shown. Data are plotted as per cent of dark control, which received no end-of-day irradiations. Controls which received only FR are represented by horizontal lines (FR). R irradiation times at 9.1 x 10-2 nE cm-2 s-' varied from I s to 50 min to give the required doses. The data for anthocyanin content of the coleoptile (A) and for mesocotyl (B) and coleoptile (C) lengths are shown. Error bars indicate SE. Data from two replicate experiments are combined. In A, each point represents
the mean of four values, two samples from each experiment.
Perkin-Elmer model 356 spectrophotometer in the split-beam mode. Four layers of Kimwipe were used as a reference approximating the light-scattering of the tissue. RESULTS Corn seedlings became thoroughly photobleached when treated with San 9789 and grown in the light. In vivo absorption spectra of single leaves from corn plants grown in the light with and without 0.2 mm San 9789 are essentially identical to those published by Jabben and Deitzer (7) for oats grown under similar conditions. At subsaturating concentrations of San 9789, the greening of the seedlings was variable. Identically treated seedlings differed in the amount of Chl they contained and also in its distribution at these intermediate concentrations. Often the leaf
GORTON AND BRIGGS
1026
tips were green while the remainder of the leaves were white; seedlings differed in how far down the leaf the green region extended. The 3-h period of imbibition in San 9789 was necessary to obtain uniform bleaching of all seedlings. The lowest concentration which would produce total bleaching was 0.1 mm. This concentration of San 9789 had no effect on growth of etiolated seedlings, but in the light the inhibitor-treated seedlings grew slightly more slowly than controls grown on water. This effect was probably caused by the limited carbohydrate supply in the treated and, therefore, nonphotosynthetic seedlings. In all further experiments, 0.1 mM San 9789 was used. Dose-response curves for decreased anthocyanin accumulation in coleoptiles and increased mesocotyl and coleoptile elongation in response to end-of-day FR irradiation of corn seedlings grown with and without San 9789 are given in Figure 1. The direction of the coleoptile response was opposite from that of etiolated grass seedlings where R caused increased coleoptile elongation and the R effect was reversed by FR (oats: refs. 3 and 12; corn: ref. 13 and W. R. Briggs, unpublished data). These results are in agreement with those of Duke et al. (4) The mesocotyl and coleoptile growth responses were not saturated, even at high doses of FR. Inhibition of anthocyanin accumulation in the coleoptiles appeared to be saturated by a FR dose of about 103 nE cm- Thresholds for all three responses for plants grown with and without San 9789 were nearly identical (log nE cm2 = 1.5). The three responses to end-of-day FR were reversible by subsequent R as indicated by the response curves shown in Figure 2. In all three cases, R caused almost complete reversal; the FR, Rtreated plants resembled control plants which were placed immediately into the dark at the end of the 8-h white-light period. This result is reasonable since the white light under which the plants were grown contained very little FR and was, therefore, functionally equivalent to R in phytochrome photoconversion. Again, all three responses showed similar dose-response characteristics and there were no significant differences in threshold (log nE cm`2 -0.5) or saturation (log nE cm-2 = 1.3) doses for plants grown with and without San 9789. DISCUSSION
Dose-response curves for induction of responses to end-of-day FR irradiations and reversal by R irradiations were essentially identical for plants grown with and without San 9789. Therefore, the phytochrome systems involved must be unaltered by the herbicide treatment. The major differences which might be expected to appear between treated and untreated seedlings are those related to the Chl content and photosynthetic capacity of the tissue. Some long-term, energy-dependent responses might not be fully expressed in achlorophyllous tissue when the carbo-
Plant Physiol. Vol. 66, 1980
hydrate supply became limiting. This limitation was not observed within the 5-day experimental period. Another type of difference which might be expected between green and achlorophyllous tissue is a change in sensitivity to R caused by differential screening of the photoreceptor by Chl (9). Chl absorbs strongly in the same spectral region as phytochrome, so one might expect increased sensitivity to R in the achlorophyllous tissue. In the experiments presented here, the dose-response curves for R reversal of the effects of end-of-day FR might be expected to be shifted to lower doses for the achlorophyllous plants. Since no such shift was seen here, screening does not seem to be an important factor for any of the responses exanmined here. Neither the coleoptiles nor the mesocotyls of light-grown seedlings contained much Chl. Although the actual site of photoreception has not been determined, it is clear that the Chl content of the photoreceptive tissue must be low or that the phytochrome must be situated in the tissue in an unscreened location. The experiments presented here were all done with constant irradiance and varied time. Results reported briefly elsewhere indicate that the reciprocity law fails for both FR and R irradiations (5). Short intense irradiations are more effective than long, dim ones. Experiments aimed at elucidating the reciprocity failure are currently underway. Regardless of irradiation conditions, however, San 9789 did not affect the ultimate responses. LITERATURE CITED 1. BARrELS, PG. A HYDE 1970 Chloroplast development in 4-chloro5(dimethylamino)-2-(a, a, a-trifluoro-m-tolyl)-3(2H)-pyridizinone (Sandoz 6706)-treated wheat seedlings. Plant Physiol 45: 807-810 2. BARrELS, PG, C MCCULLOUGH 1972 A new inhibitor of carotenoid synthesis in higher plants: 4-chloro-5(dimethylamino)-2-a, a, a-trifluoro-m-tolyl)-3(2H)pyridizinone (Sandoz 6706). Biochem Biophys Res Commun 48: 16-22 3. BLAAUW, OH. G BLAAUW-JANSEN, WF VAN LEEUWEN 1968 An irreversible redlight-induced growth response in A vena. Planta 82: 87-104 4. DUKE, SO, AW NAYLOR, JL WICKLIFF 1977 Phytochrome control of longitudinal growth and phytochrome synthesis in maize seedlings. Physiol Plant 40: 59-68 5. GoiroN, HL, WR BRIGGS 1980 Reverse reciprocity failure of phytochromemediated responses in light-grown corn. Carnegie Inst Wash Year Book. In press 6. JABBEN, M, GF DEILrzER 1978 Spectrophotometric phytochrome measurements in light-grown Avena sativa L. Planta 143: 309-313 7. JABBEN, M. GF DEILZER 1978 A method for measuring phytochrome in plants grown in white light. Photochem Photobiol 27: 799-802 8. JABBEN, M, GF DEIrZER 1979 Effects of the herbicide San 9789 on photomorphogenic responses. Plant Physiol 63: 481-485 9. Jose, AM, E. SCHAFER 1978 Distorted phytochrome action spectra in green plants. Planta 138: 25-28 10. JosE, AM. D VINCE-PRUE, JR HIL-roN 1977 Chlorophyll interference with phytochrome measurement. Planta 135: 119-123 I 1. PRA-r-r, LH 1978 Molecular properties of phytochrome. Photochem Photobiol 27: 81 105 12. SCHNEIDER, CL 1941 The effect of red light on growth of the A vena seedling with special reference to the first internode. Am J. Bot 28: 878-886 13. VANDERHOEF, LN, PH QUAIL. WR BRIGGS 1979 Red light-inhibited mesocotyl elongation in maize seedlings. Plant Physiol 63: 1062-1067
