481  2000 American Society for Photobiology 0031-8655/00 $5.00+0.00 Photochemistry and Photobiology, 2000, 71(4): 481–486 Two Photobiological Pathways of Phytochrome A Activity, Only One of Which Shows Dominant Negative Suppression by Phytochrome B Jorge J. Casal*, Marcelo J. Yanovsky and Juan P. Luppi² 1 IFEVA, Facultad de Agronomı ´a, Universidad de Buenos Aires, Buenos Aires, Argentina Received 15 November 1999; accepted 10 January 2000 ABSTRACT The plant receptor phytochrome A (phyA) mediates re- sponses like hypocotyl growth inhibition and cotyledon unfolding that require continuous far-red (FR) light for maximum expression (high-irradiance responses, HIR), and responses like seed germination that can be induced by a single pulse of FR (very-low-fluence responses, VLFR). It is not known whether this duality results from either phyA interaction with different end-point process- es or from the intrinsic properties of phyA activity. Eti- olated seedlings of Arabidopsis thaliana were exposed to pulses of FR (3 min) separated by dark intervals of dif- ferent duration. Hypocotyl-growth inhibition and coty- ledon unfolding showed two phases. The first phase (VLFR) between 0.17 and 0.5 pulses·h -1 , a plateau be- tween 0.5 and 2 pulses·h -1 and a second phase (HIR) at higher frequencies. Reciprocity between fluence rate and duration of FR was observed within phases, not between phases. The fluence rate for half the maximum effect was 0.1 and 3 mol·m -2 ·s -1 for hourly pulses of FR (VLFR) and continuous FR (HIR), respectively. Overexpression of phytochrome B caused dominant negative suppression under continuous but not under hourly FR. We conclude that phyA is intrinsically able to initiate two discrete pho- toresponses even when a single end-point process is con- sidered. INTRODUCTION The small weed Arabidopsis thaliana possesses the red light (R)‡/far-red (FR) light photoreceptors phytochrome A (phyA), B (phyB), C, D and E (1,2), the blue/UV-A pho- toreceptors cryptochromes 1 and 2 (3) and phototropin (4), and UV-B photoreceptor(s) yet unidentified in molecular terms. This repertoire of photoreceptors allows plants to *To whom correspondence should be addressed at: IFEVA, Facultad de Agronomı ´a, Universidad de Buenos Aires, Av. San Martı ´n 4453, Buenos Aires 1417, Argentina. Fax 5411-4514-8730; e-mail: casal@ifeva.edu.ar ²Current address: INGEBI, Vuelta de Obligado 2490, 1428-Buenos Aires, Argentina. ‡Abbreviations: FR, far-red light; HIR, high-irradiance response; Pfr, FR-absorbing form of phytochrome; phyA, phytochrome A; phyB, phytochrome B; R, red light; VLFR, very-low-fluence re- sponse. sense irradiance, spectral composition, light direction and photoperiod. A single pulse of either FR or very low fluences of R is often enough to induce seed germination and several de- etiolation processes (5,6). Studies with transgenic seedlings of tobacco or rice overexpressing oat phyA (7,8), and with phyA null mutant seeds of Arabidopsis (9,10) indicate that these responses are mediated by phyA. In Arabidopsis, hy- pocotyl growth inhibition, cotyledon unfolding, anthocyanin synthesis, etc. show no long-term response to a single pulse of FR but are strongly affected by continuous FR perceived by phyA (11–13). There are obvious photobiological differ- ences when different end-point processes (seed germination, hypocotyl growth, etc.) are compared. Hourly pulses of either FR or very-low fluences of R, perceived by phyA, induce some degree of hypocotyl growth inhibition and cotyledon unfolding (14). Interestingly, com- pared to the Landsberg erecta alleles, the Columbia alleles of the VLF loci (VLF1 and VLF2) severely reduce the effects of hourly FR pulses but not those of continuous FR on hy- pocotyl growth and cotyledon unfolding (15). The effects of a single FR pulse on chlorophyll synthesis and seed germi- nation are also reduced by the Columbia alleles. Thus, phyA- mediated effects of a single pulse or hourly pulses of FR can be genetically dissected from the effects of continuous FR. Based on this genetic dissection, we have proposed a model where phyA would be able to operate via two differ- ent pathways (16). The very-low-fluence response (VLFR) pathway, operative even under infrequent (e.g. hourly or less frequent) FR pulses, and the high-irradiance response (HIR) pathway, requiring continuous FR. In other words, continu- ous FR could be setting into motion processes that do not take place under pulsed FR. In this context, the HIR is mea- sured as the specific effect of continuous FR, i.e. the differ- ence between the effects of continuous and hourly FR. One of the predictions of the above hypothesis is that phyA-mediated effects on the hypocotyl growth or cotyledon unfolding should evidence two discrete components (corre- sponding to VLFR and HIR) in a photobiological analysis. The aim of the present study was to test this prediction. In addition, since overexpressed phyB interferes with phyA ac- tivity under continuous FR (17,18) the extent of dominant negative suppression caused by overexpressed phyB was also compared for VLFR and HIR. MATERIALS AND METHODS Plant material. A. thaliana of the ecotypes Landsberg erecta, Co- lumbia or Nossen were used in this work. Null mutants of phyB in