Different phototransduction kinetics of phytochrome A and phytochrome B in Arabidopsis thaliana

The kinetics of phototransduction of phytochrome A (phyA) and phytochrome B (phyB) were compared in etiolated Arabidopsis thaliana seedlings. The responses of hypocotyl growth, cotyledon unfolding, and expression of a light-harvesting chlorophyll a/b-binding protein of the photosystem II gene promoter fused to the coding region of beta-glucuronidase (used as a reporter enzyme) were mediated by phyA under continuous far-red light (FR) and by phyB under continuous red light (R). The seedlings were exposed hourly either to n min of FR followed by 60 minus n min in darkness or to n min of R, 3 min of FR (to back-convert phyB to its inactive form), and 57 minus n min of darkness. For the three processes investigated here, the kinetics of phototransduction of phyB were faster than that of phyA. For instance, 15 min R h-1 (terminated with a FR pulse) were almost as effective as continuous R, whereas 15 min of FR h-1 caused less than 30% of the effect of continuous FR. This difference is interpreted in terms of divergence of signal transduction pathways downstream from phyA and phyB.     

Expression of functional oat phytochrome A in transgenic rice

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Here, we demonstrate in vitro, using purified recombinant photoreceptors, that Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo.     

A sequence-specific RNA-binding protein complements apobec-1 To edit apolipoprotein B mRNA

The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.     

Interaction of cryptochrome 1, phytochrome, and ion fluxes in blue-light-induced shrinking of Arabidopsis hypocotyl protoplasts

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl- and K+. The postshrinking volume recovery is achieved by K+ and Cl- influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.     

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.     

A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots

Targeting of protein cargo to the vacuole/lysosome is a multistep process that appears to have conserved features between mammalian, yeast, and plant cells. In each case, some soluble vacuolar/lysosomal proteins are believed to be bound by transmembrane cargo receptors in the trans-Golgi network (TGN) that redirect these proteins into clathrin-coated vesicles. These vesicles then appear to be transported to the prevacuole/endosome by a trafficking machinery that requires components identified in other vesicle-targeting steps such as N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), SNAP receptors (SNAREs), rab-type GTPases, and Sec1p homologs. Two likely members of this trafficking machinery have been characterized from Arabidopsis thaliana: AtPEP12p, a t-SNARE that resides on a what we now call a prevacuolar compartment, and AtELP, a protein that shares many common features with mammalian and yeast transmembrane cargo receptors. Here, we have further investigated the intracellular distribution of AtELP. We have found that AtELP is located at the trans-Golgi of Arabidopsis root cells, and that its C terminus can preferentially interact in vitro with the mammalian TGN-specific AP-1 clathrin-adapter complex, suggesting a likely role in clathrin-coated, vesicle-directed trafficking at the TGN. Further, consistent with a role in trafficking of vacuolar cargo, we have found that AtELP partially colocalizes with AtPEP12p on a prevacuolar compartment.     

A pronounced thymic B cell deficiency in the spontaneously diabetic BB rat

In an attempt to elucidate the origin of the T cell lymphopenia and/or the beta-cell-specific autoimmunity observed in diabetes-prone Bio-Breeding (DP-BB) rats, a thymic cDNA library was subjected to differential screening with thymic cDNA probes of DP-BB rats and nonlymphopenic nondiabetic controls. This approach resulted in the identification of a prominent lack of thymic B cells in DP-BB rats. This deficiency is distinct from a less pronounced peripheral B cell deficiency of different timing. The thymic B cell defect is linked to the lymphopenia trait on chromosome 4 and thereby with susceptibility to diabetes in crosses involving the DP-BB rat. In conclusion, our data suggest that the contribution of thymic B cells to the (negative) selection of thymocytes is inadequate in DP-BB rats, thus providing a plausible explanation for at least some of the spontaneous autoimmune phenomena in this animal model.     

A monoclonal antibody affects chromophore apoprotein interactions of phytochrome A

A monoclonal antibody designated Mep-1 was raised against phytochrome A from pea (Pisum sativum L.). The binding of this antibody (class IgG1) to partially degraded phytochrome (114 kDa) caused a considerable increase in the far-red peak at the red-light-induced stationary state. The effect reached a plateau value when the antibody and phytochrome were present in approximately equimolar amounts. The dark transformation of the far-red-light-absorbing form to the red-light-absorbing form of the 114 kDa phytochrome was inhibited by the addition of the antibody. However, binding of the antibody to the undegraded 121 kDa phytochrome had no effects on the spectrum of the red-light-induced steady state. The site at which the antibody bound to phytochrome was determined to be between amino acid residues 256 and 383 of pea phytochrome A. This is the first report of a monoclonal antibody that enhances the far-red absorption of phytochrome in the red-light-induced photostationary state.     

New Arabidopsis cue mutants suggest a close connection between plastid- and phytochrome regulation of nuclear gene expression

We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue (chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599-1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1 and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 and cue9). For each of the mutants, the reduction in CAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception of cue6, were compromised in their ability to derepress CAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.     

A 44-month clinical-brain MRI follow-up in a patient with B12 deficiency

We report a 51-year-old woman with vitamin B12 deficiency who presented with slight megaloblastic anemia and severe neurologic deficits associated with multiple focal and confluent T2-weighted white matter hyperintensities on brain MRI. Forty-four months after initiation of hydroxocobalamin therapy, there was clinical improvement and striking reduction in the MRI abnormalities. B12 deficiency should be considered in the differential diagnosis of neurologic disorders associated with multiple areas of white matter hyperintensities on T2-weighted brain MRI.     

The amino-terminus of phytochrome A contains two distinct functional domains

Management of labour carries the responsibility of achieving safe delivery of the mother and baby, while avoiding prolonged labour and fetal distress. This requires close attention to uterine activity, which is particularly important in labours in which contractions are stimulated pharmacologically. Whether labour is induced or augmented for slow progress, the principal aim should be to make the use of oxytocin safe. Better awareness and understanding of the effects of oxytocin, aided by appropriate methods of monitoring, will minimize iatrogenic intervention and maximize the chances of normal delivery.     

The danger of B12 deficiency in the elderly

Vitamin B12 deficiency damages nerve cells and aggravates nervous system disorders even in the absence of evidence of anaemia. Prevalence of B12 deficiency increases with age especially over 65 and is frequently associated with Alzheimer's disease. Recent American surveys record a higher prevalence of B12 deficiency and of undiagnosed and untreated pernicious anaemia in the elderly than reported earlier. B12 deficiency is also reported to be a risk factor for heart disease, stroke and accelerated ageing.     

Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A

Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity. Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid 'strep-tag' peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa. This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%. Incubation of ASPHYA-ST with phytochromobilin (P phi B) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both P phi B and PCB adducts of ASPHYA-ST were photoactive--the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima. Although the PEB adduct of ASPHYA-ST was photochemically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm. The superimposability of its absorption and fluorescence excitation spectra established that a single biliprotein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB. Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources. The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.     

Dynamics of the N-terminal alpha-helix unfolding in the photoreversion reaction of phytochrome A

Time-resolved circular dichroism spectroscopy in the far-UV spectral region was used to examine the intermediates of the phytochrome photoreversion reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 micros) and meta-Fb (tau = 5.5 ms), have been identified in a simple sequential kinetic photoreversion mechanism by absorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Butler, W. L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry 11, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121-132; Eilfeld, P., & Rüdiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (1996) Biochemistry 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermediate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unfolding process occurs with a time constant of 310 +/- 125 micros, which is consistent with the 265-micros lifetime for meta-Fa.     

Physician's management of suspected vitamin B12 deficiency

A retrospective study was undertaken to audit physician's management of patients with a low serum level of vitamin B12 who were admitted to a university-affiliated teaching hospital during 1 year. Among the 34 patients 13 were proved to have pernicious anemia or vitamin B12 malabsorption, but for 12 of them there were unnecessary delays (several days or weeks) before initiation of investigation and therapy. An additional six patients, who had low serum levels of vitamin B12 and macrocytosis, most likely had true vitamin B12 deficiency, but proper investigation was not done and they did not receive any vitamin B12 or folic acid therapy. In another nine cases unexplained low serum levels of vitamin B12 were not properly investigated, and the patients either did not receive any vitamin B12 therapy or received it without proper documentation of a deficiency. Suggestions for facilitating early detection, investigation and treatment of megaloblastic anemia or vitamin B12 deficiency are given.