Light, phytochrome signalling and photomorphogenesis in Arabidopsis.

The phytochromes is a family of plant photoreceptors that control growth and development in response to environmental cues. Red and far-red light are the most efficient wavebands to induce conformational changes of phytochromes and consequently modify their kinetics, nuclear/cytoplasmic partitioning, ability to phosphorylate substrates, and physical interaction with proteins that bind DNA. Many players in phytochrome signalling have been identified and a complex, highly regulated network is envisaged. Here we describe the connection between different features of the phytochrome signalling network and the versatile relationship between light signals and physiological outputs shown by phytochromes.

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses.

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.

The VLF loci, polymorphic between ecotypes Landsberg erecta and Columbia, dissect two branches of phytochrome A signal transduction that correspond to very-low-fluence and high-irradiance responses.

Phytochromes play a key role in the perception of light signals by plants. In this study, the three classical phytochrome action modes, i.e. very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR), were genetically dissected using phyA and phyB mutants of Arabidopsis thaliana (respectively lacking phytochrome A or phytochrome B) and a polymorphism between ecotypes Landsberg erecta and Columbia. Seed germination and potentiation of greening, hypocotyl growth inhibition and cotyledon unfolding in etiolated seedlings of the ecotype Landsberg erecta showed biphasic responses to the calculated proportion of active phytochrome established by one light pulse or repeated light pulses. The first phase, i.e. the VLFR, was absent in the phyA mutant, normal in the phyB mutant (both in the Landsberg erecta background) and severely deficient in Columbia. The second phase, i.e. the LFR, was present in the phyA mutant, deficient in the phyB mutant and normal in Columbia. Under continuous far-red light, HIR of etiolated seedlings were absent in phyA and normal in phyB and Columbia. The segregation of VLFR in recombinant inbred lines derived from a cross between Landsberg erecta and Columbia was analysed by MAPMAKER/QTL. Two quantitative trait loci, one on chromosome 2 (VLF1) and another on chromosome 5 (VLF2), were identified as responsible for the polymorphism. Phytochrome A is proposed to initiate two transduction pathways, VLFR and HIR, involving different cells and/or different molecular steps. This is the first application of the analysis of quantitative trait loci polymorphic between ecotypes to dissect transduction chains of environmental signals.

A 146 bp fragment of the tobacco Lhcb1*2 promoter confers very-low-fluence, low-fluence and high-irradiance responses of phytochrome to a minimal CaMV 35S promoter.

The occurrence of very-low-fluence responses (VLFR), low-fluence responses (LFR) and high-irradiance responses (HIR) of phytochrome was investigated for the expression of the gene of beta-glucuronidase (gusA) under the control of the tobacco Lhcb1*2 promoter, in etiolated transgenic tobacco seedlings. The activity of beta-glucuronidase (GUS) showed biphasic responses to the calculated proportion of Pfr provided by light pulses. The first phase (i.e. the VLFR) showed a maximum for Pfr levels characteristic of far-red light. The second phase (i.e. the LFR) was observed at higher Pfr levels and was reversible by far-red light pulses. The strong effect of continuous far-red light (i.e. HIR) was fluence-rate-dependent and could not be replaced either by hourly pulses of the same spectral composition and total fluence or by very low fluences of red light. Deletion of the Lhcb1*2 promoter to -453 caused little loss of GUS activity. The -453 to -31, -270 to -31 and -176 to -31 fragments of the Lhcb1*2 promoter conferred proportionally normal VLFR, LFR and HIR to a truncated (-46 to +8) CaMV 35S minimal promoter. This is the first demonstration of the presence of three phytochrome action modes in the control of the transcriptional activity of a single gene. The cis-acting regulatory elements necessary for VLFR, LFR and HIR are present in a 146 bp fragment of the tobacco Lhcb1*2 promoter.

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis.

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broad-band Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R+FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.