Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Transient down-regulation of phytochrome mRNA abundance in etiolated cucumber cotyledons in response to continuous white light

Phytochrome mRNA abundance decreased to 20% of the initial level in etiolated cucumber (Cucumis sativus L.) cotyledons exposed to continuous white light. Unexpectedly, by 12 hours of continuous white light, phytochrome mRNA had reaccumulated to 60% of the control level. High stringency RNA blot analyses suggest that it is the mRNA encoding type I phytochrome that reaccumulates.     

Photophysiology of the Elongated Internode (ein) Mutant of Brassica rapa: ein Mutant Lacks a Detectable Phytochrome B-Like Polypeptide

Several phytochrome-controlled processes have been examined in etiolated and light-grown seedlings of a normal genotype and the elongated internode (ein/ein) mutant of rapid-cycling Brassica rapa. Although etiolated ein seedlings displayed normal sensitivity to prolonged far-red light with respect to inhibition of hypocotyl elongation, expansion of cotyledons, and synthesis of anthocyanin, they displayed reduced sensitivity to prolonged red light for all three of these deetiolation responses. In contrast to normal seedlings, light-grown ein seedlings did not show a growth promotion in response to end-of-day far-red irradiation. Additionally, whereas the first internode of light-grown normal seedlings showed a marked increase in elongation in response to reduced ratio of red to far-red light, ein seedlings showed only a small elongation response. When blots of protein extracts from etiolated and light-treated ein and normal seedlings were probed with monoclonal antibody to phytochrome A, an immunostaining band at about 120 kD was observed for both extracts. The immunostaining intensity of this band was substantially reduced for extracts of light-treated normal and ein seedlings. A mixture of three monoclonal antibodies directed against phytochrome B from Arabidopsis thaliana immunostained a band at about 120 kD for extracts of etiolated and light-treated normal seedlings. This band was undetectable in extracts of ein seedlings. We propose that ein is a photoreceptor mutant that is deficient in a light-stable phytochrome B-like species.     

Identification of two loci involved in phytochrome expression in Nicotiana plumbaginifolia and lethality of the corresponding double mutant

Four Nicotiana plumbaginifolia mutants exhibiting long hypocotyls and chlorotic cotyledons under white light, have been isolated from M2 seeds following mutagenesis with ethyl methane sulphonate. In each of these mutants, this partly etiolated in white light (pew) phenotype is due to a recessive nuclear mutation at a single locus. Complementation analysis indicates that three mutants, dap5, ems28 and ems3-6-34, belong to a single complementation group called pew1, while dap1 defines the pew2 locus. The mutants at pew1 contain normal levels of immunochemically detectable apoprotein of the phytochrome that is relatively abundant in etiolated seedlings, but are deficient in spectrophotometrically detectable phytochrome, whether seedlings are grown in darkness or light. Moreover, biliverdin, a precursor of the phytochrome chromophore, restores light-regulated responses in pew1 mutants and increases their level of photoreversible phytochrome when grown in darkness. These results indicate that the pew1 locus may be involved in chromophore biosynthesis. The mutant at the pew2 locus displays no photoreversible phytochrome in etiolated seedlings, but does contain normal levels of photoreversible phytochrome when grown in the light. Biliverdin had little effect on light-regulated responses in this mutant. In addition, biliverdin did not alter the level of phytochrome in etiolated seedlings. These observations lead us to propose that this mutant could be affected in the phyA gene itself. We have also obtained the homozygous double mutant at the pew1 and pew2 loci. This double mutant is lethal at an early stage of development, consistent with a critical role for phytochrome in early development of higher plants.     

Phytochrome is involved in the light-regulation of vindoline biosynthesis in catharanthus

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyl-transferase (DAT) catalyzes the final step in the biosynthesis of the monoterpenoid indole alkaloid, vindoline. Previous studies have shown that the appearance of DAT activity in etiolated seedlings of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme activity is restricted principally to the cotyledons. Evidence is now presented that phytochrome is involved in the light-mediated induction of DAT activity in Catharanthus cotyledons.     

Phytochrome Photoconversion in Vivo: Comparison between Measured and Predicted Rates

The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons.     

Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings

The diversity of abundant mRNA sequences in various parts of 4-d etiolated pea seedlings (Pisum sativum L. var. Rondo CB) was compared by a cell-free translation of the mRNAs in the presence of [³⁵S]methionine and by an analysis of the products by two-dimensional electrofocussing/ electrophoresis (2D separation). The various parts of the seedlings were also examined for the pattern of protein synthesis in vivo. Proteins were labeled by injection of [³⁵S]methionine into the cotyledons, followed by 2D separation of the products. Over 95% of the abundant mRNA sequences and newly synthesized abundant polypeptides were shared by all parts of etiolated seedlings, including the cotyledons. However, a few distinct differences were observed when comparing mRNAs of roots and shoots; the most prominent among these were a group of six abundant mRNA sequences found exclusively in shoots. Only about 30% of the polypeptides synthesized on isolated RNA could be traced in equivalent positions on the gels as the polypeptides synthesized in vivo. Analysis of total RNA from light-grown pea seedlings showed the appearance of some twenty-five translation products not found with total RNA from etiolated seedlings, while about nine other translation products disappeared. At least ten of the light-induced RNA sequences were also present after growth in low-intensity red light (>600 nm) and are therefore thought to be controlled by the phytochrome system. Comparison of 11-d light-grown pea plants with 4-d light-grown seedlings did not reveal additional translatable RNA sequences, indicating that the major morphogenetic changes that occur after 4 d are not accompanied by significant changes in the pattern of abundant RNA sequences.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.