Flowering regulation by tissue specific functions of photoreceptors

Flowering is one of the most important steps in a plant life cycle. Plants utilize light as an informational source to determine the timing of flowering. In Arabidopsis, phytochrome A (phyA), phyB and cryptochrome2 (cry2) are major photoreceptors that regulate flowering. These photoreceptors perceive light stimuli by leaves for the regulation of flowering. A leaf is an organ consisting of different tissues such as epidermis, mesophyll and vascular bundles. In the present study, we examined in which tissue the light signals are perceived and how those signals are integrated within a leaf to regulate flowering. For this purpose, we established transgenic Arabidopsis lines that expressed a phyB-green fluorescent protein (GFP) fusion protein or a cry2-GFP fusion protein in organ/tissue-specific manners. Consequently, phyB was shown to perceive light stimuli in mesophyll. By contrast, cry2 functioned only in vascular bundles. We further confirmed that both phyB-GFP and cry2-GFP regulated flowering by altering the expression of a key flowering gene, FT, in vascular bundles. In summary, perception sites for different spectra of light are spatially separated within a leaf and the signals are integrated through the inter-tissue communication.Key words: photoreceptor, light, flowering, phytochrome, cryptochrome, inter-tissue signalThe timing of flowering is strictly regulated by environmental conditions such as light. Two aspects of light, spectral nature and photoperiod, dramatically affect flowering. In Arabidopsis, phyB and phyA/cry2 are the major photoreceptors mediating these responses. Although photoreceptors are expressed in almost all organs,¹ partial irradiation and grafting analyses have demonstrated that plants perceive light signals only in leaves.^2–4 However, roles for different tissues in a leaf remained unknown due to a lack of a proper method. To answer the question, we established Arabidopsis transgenic lines that expressed phyB-GFP or cry2-GFP on the respective mutant backgrounds. The resultant transgenic lines were examined for their flowering phenotype. Consequently, we found that phyB-GFP in mesophyll but not in other tissues regulated flowering.⁵ By contrast, cry2-GFP functioned only in vascular bundles.⁶A strong genetic interaction between phyB and cry2 in the regulation of flowering is known.^7,8 Cry2 regulates the flowering by suppressing the inhibitory effect of phyB on flowering. Hence, cry2 function is observed only in the presence of phyB. Conversely, the effect of phyB is exaggerated in the cry2 mutant, because phyB is not counteracted by cry2 in its absence. Here, we tested how phyB and cry2 in different tissues regulated flowering in the absence of the other photoreceptor. For this purpose, we took a physiological approach. Phenotype of the phyB-GFP lines was examined under monochromatic red light, in which phyB but not cyr2 is activated. As expected, phyB-GFP in mesophyll but not in vascular bundles strongly affected the flowering in this condition (Fig. 1A). We also tested the cry2-GFP function when phyB was not activated. Namely, plants were placed under blue light supplemented with strong far-red light. As expected, cry2-GFP failed to affect the flowering even under this condition regardless of where it was expressed (Fig. 1B).Open in a separate windowFigure 1FT expression under phyB or cry2 inactive conditions. Total RNA was extracted from the seedlings grown under long-day condition for 10 days and subjected to qRT-PCR for FT expression analysis. Data were normalized to the level of FT mRNA in (A) of the wild type, which was set to 1 arbitrary unit (a.u.). Mean ± SE (n = 4). WT, wild type. (A) Long-day red light, (16L 8D; 10 µmol m^-2 s^-1). WT, wild type; phyB, phyB mutant; Bpro, PHYB promoter-PHYB-GFP; PBT56, phyB-GFP in mesophyll; PBT239, phyB-GFP in vascular bundles.⁵ (B) Long-day blue and far-red light (16L 8D; blue light, 3 µmol m^-2 s^-1; far-red light, 10 µmol m^-2 s^-1). WT, wild type; cry2, cry2 mutant; pCRY-C2G, CRY2 promoter-CRY2-GFP; pCAB-C2G, CAB3 promoter-CRY2-GFP; pSUC-C2G, SUC2 promoter-CRY2-GFP.⁶Photoreceptors regulate flowering by altering the expression of a key flowering regulator, FT.^9,10 Interestingly, the FT gene is expressed specifically in vascular bundles.¹¹ Indeed, mesophyll phyB-GFP controlled the expression of FT in vascular bundles. Hence, there must be a mechanism by which the light signal is transduced from mesophyll to vascular bundles to regulate the FT expression in vascular bundles. It should be noted here that FT is not the sole factor involved in the light regulation of flowering. Factors such as CO, SPA, COP1 and PFT1 are known to link the photoreceptors and FT.^12–14 These factors most likely function in leaves. However, their function sites at the tissue level remain totally unknown except for CO. The biological clock is another class of machinery that is tightly related to the light signal transduction pathway.¹⁵ Unfortunately, function sites of the clock components for the regulation of flowering remain unclear. The future work should reveal those sites. Such analyses should finally provide a complete picture illustrating a network of the inter-tissue signaling for the regulation of flowering.The present work urges us to indentify the molecule that mediates the inter-tissue signaling between mesophyll and vascular bundles. Potential candidates include phytohormones, microRNA¹⁶ and peptides.¹⁷ Among phytohormones, gibberellin promotes flowering.¹⁸ However, gibberellin is probably not the answer because gibberellin does not alter the FT expression directly. Except gibberellin, no exogenously added phythromone dramatically affects flowering in Arabidopsis. It is known that microRNA such as miR172, miR159 and miR156 are involved in the regulation of flowering time.¹⁹ However, those microRNA''s neither regulate the FT expression nor are regulated by light. Since most of microRNA''s has not been intensively studied yet, it remains possible that one of them may mediate the above inter-tissue signal. Another potential candidate is a peptide. Although not much is known about peptide hormones in plants yet, peptides such as PSK,²⁰ xylogen²¹ and CLE²² have been shown to regulate cell growth and differentiation. Although none of peptides is known to regulate flowering in plants at present, a future work may reveal a novel peptide that mediates the inter-tissue signals for flowering.     

Metabolic alternations in SOD-deficient Escherichia coli cells when cultivated under oxidative stress from photoexcited titanium dioxide

Superoxide dismutase (SOD)-deficient Escherichia coli was cultivated under the oxidative stress generated by photoexcited titanium dioxide. These cells showed higher growth rate and glucose consumption rate with accelerated accumulation of acetic acid in the medium, compared to the cells cultivated under the normal condition without the stress. Under the stress condition, the activity of acetate kinase and mRNA expressions of the enzymes for acetic acid production (pta and ackA) were approximately doubled, while the activity of citrate synthase and mRNA expressions of the enzymes in TCA cycle (gltA, acnA, icd, sucA, sucC, sdhA, fumA and mdh) were repressed by about half, as compared with those under the normal condition. These results suggest that the stress-suffering cells switch the metabolic pathway into a “suppressed aerobiosis”, possibly for lowering the generation of reactive oxygen species.     

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.     

A Monoclonal Antibody Modulates Neutrophil Adherence While Enhancing Cell Motility

A monoclonal antibody (MoAb) to human neutrophils, designated 3H9, was established by screening for the inhibition of neutrophil adherence to plastic plates containing a medium supplemented with fetal calf serum (FCS medium). The antigen recognized by 3H9 was shown to be present on human leukocytes and found at the highest levels on granulocytes. On Western blotting, 3H9 reacted with a molecule having a molecular weight of 80 kDa. When this MoAb was added at the same time as a neutrophil stimulant (fMLP), the inhibition of neutrophil adherence to plastic plates in the presence of FCS medium was observed after 60 min incubation. Furthermore, this MoAb enhanced not only fMLP-induced chemotaxis but random migration of neutrophils as well. The mechanisms of these phenomena are discussed.     

A new measure for upright stability

The control of balance is a primary objective in most human movements. In many cases, research or practice, it is essential to quantitatively know how good the balance is at a body posture or at every moment during a task. In this paper we suggest a new measure for postural upright stability which assigns a value to a body state based on the probability of avoiding a fall initiation from that state. The balance recovery problem is solved for a population sample using a strength database, and the probability of successfully maintaining the balance is found over the population and called the probability of recovery (PoR). It, therefore, describes an attribute of a body state: how possible the control of balance is, or how safe being at that state is. We also show the PoR calculated for a 3-link body model for all states on a plane, compare it to that found using a 2-link model, and compare it to a conventional metric: the margin of stability (MoS). It is shown, for example, that MoS may be very low at a state from which most of the people will be able to easily control their balance.     

A small molecule agonist of an integrin, alphaLbeta2

The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, compound 4, functions as an agonist of alpha(L)beta(2) in Ca(2+) and Mg(2+)and as an antagonist in Mn(2+). We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of alpha(L)beta(2) in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.     

Physcomitrella patens Has Kinase-LRR R Gene Homologs and Interacting Proteins

Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein–protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis.     

A new filter-eluting solution that facilitates improved recovery of Cryptosporidium oocysts from water

Cryptosporidium is a zoonotic coccidian parasite associated with diarrhea, and the disinfectant-resistant oocysts are threats to public health even in industrialized countries. In order to make an accurate assessment of the risk to public health, a detection method that has a high recovery rate of oocysts in water is required. In this study, we developed a new filter-eluting solution that facilitates more efficient recovery of Cryptosporidium oocysts from different kinds of water samples. The filter-eluting solution, referred to as PET, consists of sodium pyrophosphate (0.02%), Tween 80 (0.01%) and trisodium EDTA (0.03%). By using PET instead of conventional filter-eluting solutions, the average recovery rate significantly increased from 25.5+/-15.1% to 43.1+/-13.9% (p<0.05). The improved oocyst recovery was likely due to the increased separation of the oocysts from debris trapped on the filter membrane as well as increased capture of the oocysts by immunomagnetic beads. We recommend that PET be used as the filter-eluting solution for detection of Cryptosporidium oocysts in environmental water.     

Volatile Anesthetics,Not Intravenous Anesthetic Propofol Bind to and Attenuate the Activation of Platelet Receptor Integrin αIIbβ3

Background

In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3.

Methods

The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis.

Results

Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation.

Conclusions

Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.     

Inverse Association between Air Pressure and Rheumatoid Arthritis Synovitis

Rheumatoid arthritis (RA) is a bone destructive autoimmune disease. Many patients with RA recognize fluctuations of their joint synovitis according to changes of air pressure, but the correlations between them have never been addressed in large-scale association studies. To address this point we recruited large-scale assessments of RA activity in a Japanese population, and performed an association analysis. Here, a total of 23,064 assessments of RA activity from 2,131 patients were obtained from the KURAMA (Kyoto University Rheumatoid Arthritis Management Alliance) database. Detailed correlations between air pressure and joint swelling or tenderness were analyzed separately for each of the 326 patients with more than 20 assessments to regulate intra-patient correlations. Association studies were also performed for seven consecutive days to identify the strongest correlations. Standardized multiple linear regression analysis was performed to evaluate independent influences from other meteorological factors. As a result, components of composite measures for RA disease activity revealed suggestive negative associations with air pressure. The 326 patients displayed significant negative mean correlations between air pressure and swellings or the sum of swellings and tenderness (p = 0.00068 and 0.00011, respectively). Among the seven consecutive days, the most significant mean negative correlations were observed for air pressure three days before evaluations of RA synovitis (p = 1.7×10⁻⁷, 0.00027, and 8.3×10⁻⁸, for swellings, tenderness and the sum of them, respectively). Standardized multiple linear regression analysis revealed these associations were independent from humidity and temperature. Our findings suggest that air pressure is inversely associated with synovitis in patients with RA.