Plant Molecular Biology5: 91-101, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht Printedin the Netherlands
Phytochrome regulation of phytochrome m R N A abundance James T. Colbert, Howard P. Hershey & Peter H. Quail
Department of Botany, University of Wisconsin-Madison, Madison, WI53706, U.S.A.
Keywords: gene expression, low-abundance mRNA, mRNA quantitation, rapid regulation, regulatory photoreceptor
Summary Pure phytochrome RNA sequence synthesized in an SP6-derived in vitro transcription system has been used as a standard to quantitate phytochrome mRNA abundance in Avena seedlings using a filter hybridization assay. In 4-day-old etiolated A vena seedlings phytochrome mRNA represents ~0.1% of the total poly(A) + RNA. Irradiation of such seedlings with a saturating red-light pulse or continuous white light induces a decline in this mRNA that is detectable within 30 min and results in a 50% reduction by--60 rain and >90% reduction within 5 h. The effect of the red-light pulse is reversed, approximately to the level of the far-red control, by an immediately subsequent far-red pulse. In seedlings maintained in extended darkness after the red-light pulse, the initial rapid decline in phytochrome mRNA level is followed by a slower reaccumulation such that 50-60% of the initial abundance is reached by 48 h. White-light grown seedlings transferred to darkness exhibit a similar accumulation of phytochrome mRNA that is accelerated by removal of residual Pfr with a far-red light pulse at the start of the dark period. The data establish that previously reported phytochrome-regulated changes in translatable phytochrome mRNA levels result from changes in the physical abundance of this mRNA rather than from altered translatability.
Introduction Phytochrome is a photoreceptor that regulates plant development in response to light at all stages of the life cycle (35). The molecule is a chromoprotein with each monomer consisting of a linear tetrapyrrole chromophore covalently linked to a polypeptide (9, 23) of 120-127 kilodaltons depending on the plant species (43). The photoreceptor has two interconvertible forms: a red-absorbing form (Pr), which absorbs maximally in the red (*max = 660 nm), and a far-red-absorbing form (Pfr), which absorbs maximally in the far-red (),max = 730 nm) region of the spectrum. Photoconversion of Pr to Pfr induces a diverse array of morphogenic responses, whereas reconversion of Pfr to Pr cancels the induction of those responses. Thus Pfr is generally considered to be the active form and Pr the inactive form of the photoreceptor.
The molecular mechanism by which Pfr induces these morphogenic responses is yet to be elucidated (28, 35). However, direct evidence has recently verified the prevailing expectation that some of these responses result from phytochrome-mediated changes in gene expression. The photoreceptor has been shown to control the expression of a number of nuclear genes including those encoding the small subunit of ribulosebisphosphate carboxylase (20, 38, 40), chlorophyll a / b binding protein (13, 20, 38, 40), protochlorophyllide reductase (1, 3), rRNA (39) and several unidentified mRNA species (40). Transcriptional control ofgene expression has been demonstrated for the small subunit of ribulosebisphosphate carboxylase, protochlorophyllide reductase and chlorophyll a / b binding protein (10, 36, 37). Expression of the phytochrome gene(s) is also light-regulated, in a negative fashion, at the protein
92 (28, 30) and translatable m R N A (7, 14) levels. The irradiation-induced decline in translatable phytochrome m R N A , which after a lag o f < 3 0 min results in reduction to
Phytochrome regulation of phytochrome m R N A abundance James T. Colbert, Howard P. Hershey & Peter H. Quail
Department of Botany, University of Wisconsin-Madison, Madison, WI53706, U.S.A.
Keywords: gene expression, low-abundance mRNA, mRNA quantitation, rapid regulation, regulatory photoreceptor
Summary Pure phytochrome RNA sequence synthesized in an SP6-derived in vitro transcription system has been used as a standard to quantitate phytochrome mRNA abundance in Avena seedlings using a filter hybridization assay. In 4-day-old etiolated A vena seedlings phytochrome mRNA represents ~0.1% of the total poly(A) + RNA. Irradiation of such seedlings with a saturating red-light pulse or continuous white light induces a decline in this mRNA that is detectable within 30 min and results in a 50% reduction by--60 rain and >90% reduction within 5 h. The effect of the red-light pulse is reversed, approximately to the level of the far-red control, by an immediately subsequent far-red pulse. In seedlings maintained in extended darkness after the red-light pulse, the initial rapid decline in phytochrome mRNA level is followed by a slower reaccumulation such that 50-60% of the initial abundance is reached by 48 h. White-light grown seedlings transferred to darkness exhibit a similar accumulation of phytochrome mRNA that is accelerated by removal of residual Pfr with a far-red light pulse at the start of the dark period. The data establish that previously reported phytochrome-regulated changes in translatable phytochrome mRNA levels result from changes in the physical abundance of this mRNA rather than from altered translatability.
Introduction Phytochrome is a photoreceptor that regulates plant development in response to light at all stages of the life cycle (35). The molecule is a chromoprotein with each monomer consisting of a linear tetrapyrrole chromophore covalently linked to a polypeptide (9, 23) of 120-127 kilodaltons depending on the plant species (43). The photoreceptor has two interconvertible forms: a red-absorbing form (Pr), which absorbs maximally in the red (*max = 660 nm), and a far-red-absorbing form (Pfr), which absorbs maximally in the far-red (),max = 730 nm) region of the spectrum. Photoconversion of Pr to Pfr induces a diverse array of morphogenic responses, whereas reconversion of Pfr to Pr cancels the induction of those responses. Thus Pfr is generally considered to be the active form and Pr the inactive form of the photoreceptor.
The molecular mechanism by which Pfr induces these morphogenic responses is yet to be elucidated (28, 35). However, direct evidence has recently verified the prevailing expectation that some of these responses result from phytochrome-mediated changes in gene expression. The photoreceptor has been shown to control the expression of a number of nuclear genes including those encoding the small subunit of ribulosebisphosphate carboxylase (20, 38, 40), chlorophyll a / b binding protein (13, 20, 38, 40), protochlorophyllide reductase (1, 3), rRNA (39) and several unidentified mRNA species (40). Transcriptional control ofgene expression has been demonstrated for the small subunit of ribulosebisphosphate carboxylase, protochlorophyllide reductase and chlorophyll a / b binding protein (10, 36, 37). Expression of the phytochrome gene(s) is also light-regulated, in a negative fashion, at the protein
92 (28, 30) and translatable m R N A (7, 14) levels. The irradiation-induced decline in translatable phytochrome m R N A , which after a lag o f < 3 0 min results in reduction to
