The Presence of Both Very Low Fluence and Low Fluence Responses

We have examined phytochrome regulated changes in transcript abundance for 11 different light regulated mRNAs in developing pea buds. Fluence-response curves were measured for changes in transcript abundance in response to red light pulses in both the low and very low fluence ranges. Most trnscripts show only low fluence responses, with a threshold of approximately 10 micromoles per square meter. All of the low fluence responses are reversible by far red light. One transipt shows a very low fluence response, with a threshold ofapproximately 10' micromoles per square meter. As expected, the very low fluence response is not far red reversible and in fact can be induced by far red light. Various flnnces of red light were also used as pretreatments before transferring seedlings to continuous white light. One transcript responds to pretreatments ini the very low fluence range, several respond to pretreatments in the low fluence range (including chlorophyll a/b binding protein RNA and ribulose-1,5-bisphosphate carboxylase RNA), and several show no response to the red light under these conditions. The threshold ofthese low fluence responses is approximately 102 micromoles per square meter, one order of magnitude greater than the threshold of the low fluence responses to red Light alone. The transcripts may also be grouped by their responses to white light treatment alone. Three of the clones correspond to transcripts whose abundance decreases after a 24 hour white lght treatment. The remainder of the mRNAs increase between 2and 10-fold in response to the 24 hour white light. Excitation ofphytochrome is responsible, in part, for the white light-induced changes observed in the steady state levels ofseveral specific nuclear encoded mRNAs. This phenomenon has been reported for transcripts of the genes encoding the cab' in barley (1), Lemna (26), pea (16, 17, 25), and mung bean (25); rbcS in Lemna (26), pea (16, 17, 25), and mung bean (25); phytochrome in oat (9); NADPH:Pchlide oxidoreductase in barley (2); and a 17 kD chloroplast polypeptide in pea (21). Recently our laboratory has demonstrated phytochrome control ofsteady state levels for 10 other transcripts in pea and four other transcripts in mung ' Supported by United States Department of Agriculture grant 78, 5921141-009-1. This is Carnegie Institute of Washington Department of Plant Biology publication 866. 2 Abbreviations: cab, Chl a/b binding protein; rbc S, the small subunit of ribulose1,-5-bisphosphate carboxylase; R, red light; FR, far-red light, LF, low fluence; VLF, very low fluence. bean (25). In Lemna it has been shown that phytochrome affects transcription of the cab and rbcS genes in isolated nuclei (23). Phytochrome regulation occurs over two fluence ranges of R (5, 7, 20). The LF response has a threshold of approximately 10' ,umol m-2 and is fully reversible by FR. This is the common phytochrome response observed in most plants. The VLF response has a threshold ofapproximately 10-3 gmol m-2. It is not reversible by FR; indeed it is induced by most FR sources (5, 7, 20). To date, the VLF response has only been observed in a limited number ofsystems. These include growth rates ofetiolated Avena coleoptiles and mesocotyls (3, 20), anthocyanin synthesis in mustard seedlings (4), and germination of dormant lettuce seeds (6, 7, 24). A VLF response for Chl accumulation in peas is apparent if R of varying fluence is used as a pretreatment followed by a dark period and a subsequent white light treatment (18, 22). Recently, we have also demonstrated, using etiolated pea buds, that cab transcripts have both a VLF and a LF response, whereas rbcS transcripts exhibit only a LF response (17). The mechanism whereby phytochrome regulates transcription and/or transcript abundance is unknown. In this paper, we describe the basic characteristics of phytochrome control for 11 unidentified phytochrome regulated messages and further characterize the phytochrome responses of cab and rbcS transcripts. We have measured the fluence-dependent accumulation of these transcripts in response to single pulses of R, the ability ofFR to reverse these R effects, and the ability ofFR to induce transcript accumulation in the absence of prior R treatments. We have also used a pretreatment protocol similar to that described above which demonstrates an effect ofVLF R for Chl accumulation in a subsequent white light period (18, 22). For several transcripts, significant differences are apparent between responses seen in these potentiation-type experiments and responses to the R pulse alone. MATERIALS AND METHODS Plant Material and Light Treatments. Seeds ofPisum sativum L. cv Alaska (W. Atlee Burpee Co., Warminster, PA) were imbibed for 5 h and grown on two layers ofwater-soaked Kimpak (Kimberly-Clarke, Roswell, GA) at 27°C, 85% RH, in absolute darkness. Plants were grown in the above conditions for 6 d, given the various light treatments, and returned to the dark for an additional 24 h after which they were either harvested or given a 24-h white light treatment. Protocols used for the various light treatments are illustrated in Figure 1. For studies of R and FR fluence dependence, groups of approximately 100 6-d-old seedlings were irradiated with single pulses of varying fluence of either R or FR and then returned to darkness for 24 h before 388 www.plantphysiol.org on July 23, 2017 Published by Downloaded from Copyright © 1985 American Society of Plant Biologists. All rights reserved. LOW AND VERY LOW FLUENCE RESPONSES FOR SPECIFIC mRNAs IN PEA