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The vibrational changes occurring in the full-length Arabidopsis cryptochrome 1 (CRY 1) protein upon illumination with blue light are investigated by Fourier transform infrared (FTIR) and ultraviolet (UV)/vis difference spectroscopy. The reversible formation of a neutral flavin radical by blue light is accompanied by changes in secondary structure of the apoprotein and this analysis is useful for FTIR studies on flavoprotein radicals in general and will facilitate future work on the cryptochrome signaling process.

Members of the cryptochrome/photolyase family ( CPF) of proteins utilize noncovalently bound light-absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His6-tagged protein expressed in Escherichia coli, that is , flavin adenine dinucleotide and N5,N10-methenyltetrahydrofolate. This demonstrates that the heterologously expressed cryptochrome 3, characterized in a number of previous studies, is a valid surrogate of the corresponding protein expressed in plants. To our knowledge, this is also a first conclusive analysis of cofactors bound to an Arabidopsis protein belonging to the CPF and purified from plant tissue. [ABSTRACT FROM AUTHOR]

Proteins from the cryptochrome/photolyase family utilize UV-A, blue or even red light to achieve such diverse functions as repair of DNA lesions by photolyases and signaling by cryptochromes. DASH-type cryptochromes retained the ability to repair cyclobutane pyrimidine dimers ( CPDs) in single-stranded DNA regions in vitro. However, most organisms possess conventional CPD photolyases responsible for repair of these lesions in vivo. Recent work showed that the DASH-type cryptochrome Cry D plays a regulatory role in diverse light-dependent processes in Fusarium fujikuroi. Here, we report our in vitro studies on heterologously expressed Ff Cry D. The purified protein contains N5, N10-methenyltetrahydrofolate and flavin adenine dinucleotide as cofactors. Photoreduction and DNA photorepair experiments confirmed that Ff Cry D is active in light-driven electron transfer processes. However, the protein showed comparable affinities for CPD-comprising and undamaged DNA probes. Surprisingly, after purification, full-length Ff Cry D as well as a truncated version containing only the PHR domain bound RNA which influenced their behavior in vitro. Moreover, binding of Ff Cry D to RNA indicates a putative role in RNA metabolism or in posttranscriptional control of gene expression. [ABSTRACT FROM AUTHOR]

Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated. [ABSTRACT FROM AUTHOR]

Examines tissue-specific and light-dependent changes of chromatin organization in barley or Hordeum vulgare. DNase I sensitivity of the nuclear genes encoding the NADPH-protochlorophyllide oxidoreductase; Light-harvesting chlorophyll a/b protein; Assay in chromatin of etiolated and green leaves and endosperm tissue of barley.

In carotenoid-deficient albina mutants of barley and in barley plants treated with the herbicide Norfiurazon the light-dependent accumulation of the mRNA for the light-harvesting chlorophyll a/b protein (LHCP) is blocked. Thus, the elimination of a functional chloroplast, either as a result of mutation or as a result of herbicide treatment, can lead to the specific suppression of the expression of a nuclear gene encoding a plastid-localized protein. These results confirm and extend earlier observations on maize [Mayfield and Taylor (1984) Eur. J. Biochem. 144, 79-84]. The inhibition of mRNA accumulation appears to be specific for the LHCP; the mRNAs encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the NADPH : protochlorophyllide oxidoreductase are relatively unaffected. The failure of the albina mutants and of Norfiurazon-treated plants to accumulate the LHCP mRNA is not exclusively caused by an instability of the transcript but rather by the inability of the plants to enhance the rate of transcription of the LHCP genes during illumination. Several chlorophyll-deficient xantha mutants of barley, which are blocked after protoporphyrin IX or Mg-protoporphyrin, and the chlorophyll-b-less mutant chlorina f2 accumulate the LHCP mRNA to almost normal levels during illumination. Thus, if any of the reactions leading to chlorophyll formation is involved in the control of LHCP mRNA accumulation it should be one between the formation of protochiorophyllide and the esterification of chlorophyllide a. While the nature of the regulatory factor(s) has not been identified our results suggest that, in addition to phytochrome (Pfr), plastid-dependent factors are required for a continuous light-dependent transcription of nuclear genes encoding the LHCP.

The transcriptional rates of four different genes in shoots of barley grown under different light regimes were quantified by monitoring nuclear RNA transcripts using gene-specific hybridization probes. Isolated nuclei were pulse-labelled with [α-32]UTP and the relative rates of light-harvesting chlorophyll a/b protein (LHCP) mRNA, NADPH:protochlorophyllide oxidoreductase mRNA, B1 bordein mRNA, and 26-S rRNA synthesis were measured. Irradiation of dark-grown plants with a red light pulse increased the rate of LHCP mRNA synthesis tenfold within 3 h, and the rate of rRNA synthesis more than twofold within 9 h. The relative rate of synthesis of the oxidoreductase mRNA decreased following a red light pulse reaching a minimum after 3–6 h. As a direct proof of phytochrome involvement in the light-induced stimulation of LHCP and the repression of the oxidoreductase transcripts for both responses, red/far-red reversibility could be demonstrated. We conclude that phytochrome is able both to increase the transcription of certain nuclear genes and decrease the transcription of others. [ABSTRACT FROM AUTHOR]

During the light-dependent transformation of etioplasts to chloroplasts a rapid decrease of the NADPH-protochlorophyllide oxidoreductase is induced. At the same time the mRNA activity coding for this enzyme protein also declines rapidly under the influence of phytochrome (Pf J. On the other hand the apoprotein of the light-harvesting chlorophyll a/b protein and its mRNA activity are inversely affected by the same photoreceptor. Cloned cDNA sequences which are complementary to these mRNAs have been used to assess the effect of phytochrome (Pfr) on the concentration of the two transcripts. The phytochrome-induced changes of the two translatable mRNAs are paralleled by corresponding changes in the steady-state concentration of the mRNA sequences. This inverse relationship between the light-dependent regulation of the NADPH-protochlorophyllide oxidoreductase and the light-harvesting chlorophyll a/b protein suggests that a single reversible triggering event can increase the transcription of certain genes and decrease the transcription of others. [ABSTRACT FROM AUTHOR]

In barley seedlings grown in the dark large amounts of thionin-specific mRNAs are present, the concentration of which rapidly declines once the seedling is exposed to light. This rapid light effect is mediated by a complex interaction of possibly two photoreceptors, phytochrome and a blue-light-absorbing photoreceptor. Parallel to the decline in mRNA content, the de novo synthesis of leaf-specific thionins ceases rapidly upon illumination of etiolated seedlings. However, thionins which have accumulated before the onset of illumination remain stable within the seedling at high concentrations. In younger leaves of mature, nonstressed barley plants grown under a 16-h-light/8-h-dark cycle thionins are still present, although at much lower concentrations. In these plants, synthesis and accumulation of thionins occur predominantly in the meristematic zone at the leaf basis, which is shielded from light through the sheath of the preceding leaf. In mature light-adapted barley plants, mRNA encoding leaf-specific thionins may reaccumulate if these plants are exposed to pathogens or other stresses. Thus, the inhibitory effect of light on the biosynthesis of thionins may be overruled by stress- and pathogen-induced signals. [ABSTRACT FROM AUTHOR]