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.     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

Multiple phytochromes are involved in red-light-induced enhancement of first-positive phototropism in Arabidopsis thaliana.

The amplitude of phototropic curvature to blue light is enhanced by a prior exposure of seedlings to red light. This enhancement is mediated by phytochrome. Fluence-response relationships have been constructed for red-light-induced enhancement in the phytochrome A (phyA) null mutant, the phytochrome B- (phyB) deficient mutant, and in two transgenic lines of Rabidopsis thaliana that overexpress either phyA or phyB. These fluence-response relationships demonstrate the existence of two response in enhancement, a response in the very-low-to-low-fluence range, and a response in the high-fluence range. Only the response in the high-fluence range is present in the phyA null mutant. In contrast, the phyB-deficient mutant is indistinguishable from the wild-type parent in red-light responsiveness. These data indiacate that phyA is necessary for the very-low-to-low but not the high-influence response, and that phyB is not necessary for either response range. Based on these results, the high-fluence response, if controlled by a single phytochrome, must be controlled by aphytochorme other than phyA of phyB. Overexpression of phyA has a negative effect and overexpression of phyB has an enhancing effect in the high-fluence range. These results suggest that overexpression of either phytochrome perturbs the function of the endogenous photoreceptor system in an unpredictable fashion.     

Interaction of phytochromes A and B in the control of de-etiolation and flowering in pea

The interactions of phytochrome A (phyA) and phytochrome B (phyB) in the photocontrol of vegetative and reproductive development in pea have been investigated using null mutants for each phytochrome. White-light-grown phyA phyB double mutant plants show severely impaired de-etiolation both at the seedling stage and later in development, with a reduced rate of leaf production and swollen, twisted internodes, and enlarged cells in all stem tissues. PhyA and phyB act in a highly redundant manner to control de-etiolation under continuous, high-irradiance red light. The phyA phyB double mutant shows no significant residual phytochrome responses for either de-etiolation or shade-avoidance, but undergoes partial de-etiolation in blue light. PhyB is shown to inhibit flowering under both long and short photoperiods and this inhibition is required for expression of the promotive effect of phyA. PhyA is solely responsible for the promotion of flowering by night-breaks with white light, whereas phyB appears to play a major role in detection of light quality in end-of-day light treatments, night breaks and day extensions. Finally, the inhibitory effect of phyB is not graft-transmissible, suggesting that phyB acts in a different manner and after phyA in the control of flower induction.     

Identification of cDNA clones encoding different domains of the basement membrane heparan sulfate proteoglycan

We have used antibodies to the basement membrane proteoglycan to screen lambda gt11 expression vector libraries and have isolated two cDNA clones, termed BPG 5 and BPG 7, which encode different portions of the core protein of the heparan sulfate basement membrane proteoglycan. These clones hybridize to a single mRNA species of approximately 12 kilobases. Amino acid sequences obtained on peptides derived from protease digests of the core protein were found in the deduced sequence, confirming the identity of these clones. BPG 5 spanned 1986 base pairs and has an open reading frame of 662 amino acids. The amino acid sequence deduced from BPG 5 contains two cysteine-rich domains and two internally homologous domains lacking cysteine. The cysteine-rich domains show homology to the cysteine-rich domains of the laminin chains. A globule-rod structure, similar to that of the short arms of the laminin chains, is proposed for this region of the proteoglycan. The other clone, BPG 7, is 2193 base pairs long and has an open reading frame of 731 amino acids. The deduced sequence contains eight internal repeats with 2 cysteine residues in each repeat. These repeats show homology to the neural-cell adhesion molecule N-CAM and the plasma alpha 1B-glycoprotein. Looping structures similar to these proteins and to other proteins of the immunoglobulin gene superfamily are proposed for this region of the proteoglycan. The sequence DSGEY was found four times in this domain and could be heparan sulfate attachment sites.     

The system of phytochromes: photobiophysics and photobiochemistry in vivo.

Phytochrome is a key photoregulation pigment in plants which determines the strategy of their development throughout their life cycle. The major achievement in the recent investigations of the pigment is the discovery of its structural and functional heterogeneity: existence of a family of phytochromes (phyA-phyE) differing by the apoprotein was demonstrated. We approach this problem by investigating the chromophore component of the pigment with the use of the developed method of in vivo low-temperature fluorescence spectroscopy of phytochrome. In etiolated plants, phytochrome fluorescence was detected and attributed to its red-light absorbing form (Pr) and the first photoproduct (lumi-R), and a scheme of the photoreaction in phytochrome, a distinction of which is the activation barrier in the excited state, was put forward. It was found that the spectroscopic and photochemical characteristics of Pr depend on the plant species and phytochrome mutants and overexpressors used, on localization of the pigment in organs and tissues, plant age, effect of preillumination and other physiological factors. This variability of the parameters was interpreted as the existence of at least two phenomenological Pr populations, which differ by their spectroscopic characteristics and activation parameters of the Pr --> lumi-R photoreaction (in particular, by the extent of the Pr --> lumi-R photoconversion at low temperatures, gamma1): the longer-wavelength major and variable by its content in plant tissues Pr' with gamma1 = 0.5 and the shorter-wavelength minor relatively constant Pr" with gamma1 < or = 0.05. The analysis of the phytochrome mutants and overexpressors allows a conclusion that phytochrome A (phyA), which dominates in etiolated seedlings, is presented by two isoforms attributed to Pr' and Pr" (phyA' and phyA", respectively). Phytochrome B (phyB) accounts for less than 10% of the total phytochrome fluorescence and belongs to the Pr" type. It is also characterized by the relatively low extent of the Pr photoconversion into the far-red-light absorbing physiologically active phytochrome form, Pfr. Fluorescence of the minor phytochromes (phyC-phyE) is negligible. The recently discovered phytochrome of the cyanobacterium Synechocystis also belongs to the phenomenological Pr" type. PhyA' is a light-labile and soluble fraction, while phyA" is a relatively light-stable and, possibly, membrane (protein)-associated. Experiments with transgenic tobacco plants overexpressing full-length and C- and N-terminally truncated oat phytochrome A suggest that phyA' and phyA" might differ by the post-translational modification of the small N-terminal segment (amino acid residues 7-69) of the pigment. PhyA' is likely to be active in the de-etiolation processes while phyA" together with phyB, in green plants as revealed by the experiments on transgenic potato plants and phytochrome mutants of Arabidopsis and pea with altered levels of phytochromes A and B and modified phenotypes. And finally, within phyA', there are three subpopulations which are, possibly, different conformers of the chromophore. Thus, there is a hierarchical system of phytochromes which include: (i) different phytochromes; (ii) their post-translationally modified states and (iii) conformers within one molecular type. Its existence might be the rationale for the multiplicity of the photoregulation reactions in plants mediated by phytochrome.     

Over-expression of a C-terminal region of phytochrome B

In an attempt to identify domains directly involved in the signal transduction of phytochrome B (phyB), we over-expressed the achromophoric C-terminal half of phyB under control of the CaMV-35S promoter in transgenicArabidopsis. In three independent transgenic lines, we detected accumulation of the introduced protein of predicted size at levels higher than that of the endogenous phyB by immunoblot analysis. Although these transgenic plants did not show any phenotype in the dark, enhancement of the phyB-dependent inhibition of hypocotyl elongation and reduction of the phytochrome A (phyA)-dependent inhibition were observed.     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

Distinct and cooperative functions of phytochromes A, B, and C in the control of deetiolation and flowering in rice

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.     

Phytochrome gene expression and phylogenetic analysis in the short-day plant Pharbitis nil (Convolvulaceae): Differential regulation by light and an endogenous clock

To investigate the role of distinct phytochrome pools in photoperiodic timekeeping, we characterized four phytochrome genes in the short-day plant Pharbitis nil. Each PHY gene had different photosensory properties and sensitivity to night break that inhibits flowering. During extended dark periods, PHYE, PHYB, and PHYC mRNA accumulation exhibited a circadian rhythmicity indicative of control by an endogenous clock. Phylogenetic analysis recovered four clades of angiosperm phytochrome genes, phyA, phyB, phyC, and phyE. All except the phyE clade included sequences from both monocots and eudicots. In addition, phyA is sister to phyC and phyE sister to phyB, with gymnosperm sequences sister to either the phyA-phyC clade or to the phyB-phyE clade. These results suggest that a single duplication occurred in an ancestral seed plant before the divergence of extant gymnosperms from angiosperms and that two subsequent duplications occurred in an ancestral angiosperm before the divergence of monocots from eudicots. Thus in P. nil, a multigene family with different patterns of mRNA abundance in light and darkness contributes to the total phytochrome pool: one pool is light labile (phyA), whereas the other is light stable (phyB and phyE). In addition, PHYC mRNA represents a third phytochrome pool with intermediate photosensory properties.     

Overexpression of Arabidopsis phytochrome B inhibits phytochrome A function in the presence of sucrose

Overexpression of phytochrome B (phyB) in Arabidopsis has previously been demonstrated to result in dominant negative interference of phytochrome A (phyA)-mediated hypocotyl growth inhibition in far-red (FR) light. This phenomenon has been examined further in this study and has been found to be dependent on the FR fluence rate and on the availability of metabolizable sugars in the growth medium. Poorly metabolized sugars capable of activating the putative hexokinase sensory function were not effective in eliciting the phytochrome interference response. Overexpressed phyB lacking the chromophore-binding site was also effective at inhibiting the phyA response, especially at higher fluence rates of FR. Overexpressed phyB produces the dominant negative phenotype without any apparent effect on phyA abundance or degradation. It is possible that phyA and phyB interact with a common reaction partner but that either the energy state of the cell or a separate sugar-signaling mechanism modulates the phytochrome-signaling interactions.     

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.     

Antagonistic but complementary actions of phytochromes A and B allow seedling de-etiolation.

Using dichromatic radiation, we show that the actions of phytochromes A and B (phyA and phyB) in Arabidopsis thaliana are antagonistic in mediating red and far-red radiation effects on seedling de-etiolation and yet act in a complementary manner to regulate de-etiolation, irrespective of spectral composition. At low phytochrome photoequilibria inhibition of hypocotyl extension was strong, because of the action of a far-red high-irradiance response mediated by phyA. At high phytochrome photoequilibria inhibition of hypocotyl extension was also strong, because of the action of phyB. At intermediate photoequilibria hypocotyl inhibition was less strong. In their natural environment, this dual action will strongly retard hypocotyl growth and promote cotyledon opening and expansion both in open daylight and under dense vegetation. Overlapping action by phyA and phyB will substantially promote de-etiolation in sparse vegetation. The antagonistic and complementary actions of phyA and phyB, therefore, allow the optimum regulation of seedling growth after emergence from the soil.