Cryptochrome blue-light photoreceptors of Arabidopsis implicated in phototropism

Phototropism-bending towards the light-is one of the best known plant tropic responses. Despite being reported by Darwin and others over a century ago to be specifically under the control of blue light, the photoreceptors mediating phototropism have remained unknown. We have characterized a blue-light photoreceptor from Arabidopsis, named CRY1 for cryptochrome 1; this photoreceptor is a flavoprotein that mediates numerous blue-light-dependent responses. In Arabidopsis, HY4 (the gene encoding CRY1) is a member of a small gene family that also encodes a related photoreceptor, CRY2, which shares considerable functional overlap with CRY1. Here we report that mutant plants lacking both the CRY1 and the CRY2 blue-light photoreceptors are deficient in the phototropic response. Transgenic Arabidopsis plants overexpressing CRY1 or CRY2 show enhanced phototropic curvature. We conclude that cryptochrome is one of the photoreceptors mediating phototropism in 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.     

NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.     

Autosomal dominant medullary cystic disease: a disorder with variable clinical pictures and exclusion of linkage with the NPH1 locus

BACKGROUND: The nephronophthisis-medullary cystic disease (NPH/MCD) complex represents a heterogeneous group of hereditary tubulointerstitial nephritis. The most common variant is juvenile recessive NPH, for which a gene locus (NPH1) has been mapped on chromosome 2q13. MCD is a less common dominant condition usually recognized later in life, which resembles NPH in many aspects, still presenting remarkable clinical differences. Nothing is known about the chromosome locus of MCD. METHODS: Five MCD families were studied. Diagnosis was made by inference from family history, type of inheritance, clinical signs and histology. Multipoint linkage analysis was performed by markers D2S293, D2S340 and D2S160 spanning the entire NPH1 locus. RESULTS: Diagnosis of MCD was made in 28 affected members (16 males; 12 females), belonging to five families. Histological diagnosis was available in 10 patients; clinical diagnosis in 11; seven deceased relatives had diagnosis of chronic nephritis. The age at diagnosis ranged from 8 to 65 years. Renal medullary cysts were found in a minority of patients. In family 1, the disease was associated with hyperuricaemia and gouty arthritis. Progression of renal disease presented intra- and extra-family variability with members of the same family showing mild elevation of creatinine or terminal renal failure. The NPH1 locus associated to recessive NPH was excluded from linkage to the dominant MCD. CONCLUSIONS: MCD might be more common than previously assumed. Variability in clinical presentation and absence of histopathological hallmarks contribute to make the diagnosis uncommon. The most remarkable clinical difference with NPH is the age of onset in some kindreds and a delayed progression towards renal failure. The exclusion of linkage to the NPH1 locus suggests the existence of an MCD responsible locus, still to be mapped.     

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

Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, represents a type of genomic instability frequently detected in many types of cancers. However, the involvement of MSI in non-Hodgkin's lymphomas (NHL) has not been conclusively investigated. In this study, we have tested the presence of MSI in 69 cases of B-cell NHL (B-NHL) representative of the various histologic categories of the disease and including 17 cases of acquired immunodeficiency syndrome (AIDS)-related B-NHL (AIDS-NHL). In addition, for selected B-NHL cases, consecutive samples obtained before and after clinical progression (with and without concomitant histologic transformation) were also investigated. Five distinct microsatellite repeats (2 dinucleotide, 2 trinucleotide, and 1 tetranucleotide repeats) were analyzed by polymerase chain reaction in all cases. MSI, defined by the presence of microsatellite alterations in two or more of the five microsatellite loci tested, was not found in NHL. In contrast to a previous study reporting the frequent association between MSI and AIDS-NHL, we found this abnormality in only 1 of 17 cases of AIDS-NHL representative of the major subtypes. Overall, these data indicate that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of B-NHL.     

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and M?ssbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.     

cot1: a regulator of Arabidopsis trichome initiation

In Arabidopsis, the timing and spatial arrangement of trichome initiation is tightly regulated and requires the activity of the GLABROUS1 (GL1) gene. The COTYLEDON TRICHOME 1 (COT1) gene affects trichome initiation during late stages of leaf development and is described in this article. In the wild-type background, cot1 has no observable effect on trichome initiation. GL1 overexpression in wild-type plants leads to a modest number of ectopic trichomes and to a decrease in trichome number on the adaxial leaf surface. The cot1 mutation enhances GL1-overexpression-dependent ectopic trichome formation and also induces increased leaf trichome initiation. The expressivity of the cot1 phenotype is sensitive to cot1 and 35S::GL1 gene dosage, and the most severe phenotypes are observed when cot1 and 35S::GL1 are homozygous. The COT1 locus is located on chromosome 2 15.3 cM north of er. Analysis of the interaction between cot1, try, and 35S::GL1 suggests that COT1 is part of a complex signal transduction pathway that regulates GL1-dependent adoption of the trichome cell fate.     

Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism

The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.     

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.     

NDR1, a pathogen-induced component required for Arabidopsis disease resistance

Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific corresponding avirulence genes. In Arabidopsis thaliana, resistance to both bacterial and fungal pathogens, mediated by several R gene products, requires the NDR1 gene. Positional cloning was used to isolate NDR1, which encodes a 660-base pair open reading frame. The predicted 219-amino acid sequence suggests that NDR1 may be associated with a membrane. NDR1 expression is induced in response to pathogen challenge and may function to integrate various pathogen recognition signals.     

Evidence for photoreceptor changes in patients with diabetic retinopathy

PURPOSE: To determine whether the rod and cone photoreceptors are affected in patients with diabetic retinopathy. METHODS: Twelve patients with diabetes and varying levels of retinopathy and nine age-similar control observers participated in this study. Two-color (500 versus 650 nm) dark-adapted thresholds were measured as a function of retinal eccentricity. Full-field flash electroretinograms were obtained using brief, high-intensity flashes. Dark-adapted rod-isolated (Wratten 47B filter) and light-adapted cone-isolated (Wratten 26 filter) electroretinographic responses were measured as a function of flash intensity. The a-wave data were fitted with a model based on photopigment transduction to obtain values for the parameters of Rmax (the maximal response) and log S (sensitivity). Standard clinical 30-Hz flicker electroretinographic responses were also measured. RESULTS: Psychophysically measured dark-adapted thresholds were elevated primarily at eccentricities of 5 degrees and 10 degrees from the fovea. Analysis of rod and cone a-wave data showed that Rmax was normal in most of the patients, but log S was reduced. Analysis of b-wave and oscillatory potential parameters showed rod and cone postreceptoral abnormalities, including changes in the rod-isolated semisaturation constant (log k), cone-mediated 30-Hz flicker, and cone-isolated oscillatory potentials. The electrophysiological results were not significantly correlated with blood glucose or glycosylated hemoglobin level. CONCLUSIONS: The results provide evidence for rod and cone receptoral and postreceptoral deficits in patients with diabetic retinopathy. The photoreceptor changes are primarily in the log S (sensitivity) parameter and are attributed to transduction abnormalities.     

Association of spectrin with a subcompartment of the endoplasmic reticulum in honeybee photoreceptor cells

In order to investigate the mechanisms involved in originating a diverse TCR repertoire in human peripheral blood we analyzed TCRV beta surface expression in different T cell subsets of unrelated individuals. The relative frequencies of 11 distinct V beta chains were determined for immature double positive (DP) as well as for mature CD4 single positive (4SP) and CD8 single positive (8SP) thymocytes, respectively. By comparing these data with expression in peripheral blood T lymphocytes of the same donors we were able to show that usage of TCRV beta in peripheral T cells is significantly (p < 0.001) depending on the pattern in mature SP thymocytes whereas the frequency of TCRV beta families in immature DP thymocytes has no impact (p > 0.2). No association with distinct HLA-haplotypes was observed. Preferential usage of V beta-families in either CD4- or CD8-positive peripheral T cells also correlates with the status in mature thymic precursors (p < 0.001). Altogether, this first combined study of TCR frequencies within different stages of human T cell ontogeny indicates that TCRV beta repertoire is determined mainly through selectional processes within the thymus. Since neither genomically imposed expression nor modulating events in the periphery seem to have strong influence on the relative expression of TCRV beta chains these findings have to be considered in future studies of human diseases.     

Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase

Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.     

COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility

The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.     

Chlamyrhodopsin represents a new type of sensory photoreceptor

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.     

PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway

Mutants in the PRT1 gene of Arabidopsis thaliana are impaired in the degradation of a normally short-lived intracellular protein that contains a destabilizing N-terminal residue. Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway. The chromosomal position of PRT1 was determined, and the PRT1 gene was isolated by map-based cloning. The 45-kDa PRT1 protein contains two RING finger domains and one ZZ domain. No other proteins in databases match these characteristics of PRT1. There is, however, a weak similarity to Rad18p of Saccharomyces cerevisiae. The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of PRT1 in the plant N-end rule pathway.     

AGO1 defines a novel locus of Arabidopsis controlling leaf development

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

Objective assessment of photoreceptor displacement and metamorphopsia: a study of macular holes

OBJECTIVE: To determine gastric secretory responses in horses treated with histamine and to determine the dose of histamine needed to elicit maximal gastric secretion. ANIMALS: 6 adult horses with an indwelling gastric cannula. PROCEDURE: Gastric contents were collected in 15-minute periods, and volume, pH, hydrogen ion concentration, hydrogen ion output, sodium concentration, and sodium output were determined. Values were determined without any treatment (baseline), after administration of pyrilamine maleate (1 mg/kg of body weight, i.v., given during a 15-minute period), and during 1-hour infusions of histamine at 3 rates (7.5, 15, and 30 microg/kg/h, i.v.). RESULTS: Volume and hydrogen ion concentration of gastric contents and hydrogen ion output were significantly increased, compared with baseline values, during histamine infusion. Mean hydrogen ion concentration and hydrogen ion output were significantly greater during infusion of histamine at a rate of 15 or 30 microg/kg/h than at a rate of 7.5 microg/kg/h. Sodium concentration was significantly decreased, compared with baseline value, during histamine infusion, but sodium output was unchanged. CONCLUSIONS: Histamine at doses of 15 and 30 microg/kg/h, i.v. stimulated maximal gastric secretion in horses. Histamine appeared to induce only parietal secretion. CLINICAL RELEVANCE: This study provides additional information related to equine gastric physiology, which may benefit further understanding of the pathogenesis of peptic ulcer disease.