bluuebunny/crossref_metadata_2025 huggingface dataset:
Split target specificity of ResT: a design for protein delivery, site selectivity and regulation of enzyme activity?
Authors: Jayaram Makkuni
Published: May 2007

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N‐terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N‐ and C‐terminal domains targeting distinct regions of the telomere. This bi‐specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.
Submicromolar hydrogen peroxide disrupts the ability of Fur protein to control free‐iron levels in Escherichia coli
Authors: Varghese Shery , Wu Amy , Park Sunny , Imlay Karin R. C. , Imlay James A.
Published: May 2007

In aerobic environments, mutants of Escherichia coli that lack peroxidase and catalase activities (Hpx–) accumulate submicromolar concentrations of intracellular H2O2. We observed that in defined medium these strains constitutively expressed members of the Fur regulon. Iron‐import proteins, which Fur normally represses, were fully induced. H2O2 may antagonize Fur function by oxidizing the Fur:Fe2+ complex and inactivating its repressor function. This is a potential problem, as in iron‐rich environments excessive iron uptake would endanger H2O2‐stressed cells by accelerating hydroxyl‐radical production through the Fenton reaction. However, the OxyR H2O2‐response system restored Fur repression in iron‐replete Luria–Bertani medium by upregulating the synthesis of Fur protein. Indeed, when the OxyR binding site upstream of fur was disrupted, Hpx– mutants failed to repress transporter synthesis, and they exhibited high levels of intracellular free iron. Mutagenesis and bacteriostasis resulted. These defects were eliminated by mutations or chelators that slowed iron import, confirming that dysregulation of iron uptake was the root problem. Thus, aerobic organisms must grapple with a conundrum: how to monitor iron levels in oxidizing environments that might perturb the valence of the analyte. The induction of Fur synthesis by the OxyR response comprises one evolutionary solution to that problem.
Cwp2p, the plasma membrane receptor for Pichia membranifaciens killer toxin
Authors: Santos Antonio , San Mauro Manuel , Abrusci Concepción , Marquina Domingo
Published: May 2007

PMKT is a channel‐forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2Δ mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT‐affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring‐defective mutants were much less sensitive to PMKT, as were wild‐type protoplasts pretreated with phosphatidylinositol‐specific phospholipase C to remove GPI‐anchored proteins, indicating that the GPI‐anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein‐entrapped liposomes containing a purified GFP–Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein‐free liposomes. Cwp2p and its GPI‐anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.
Mechanism of cell surface expression of the Streptococcus mitis platelet binding proteins PblA and PblB
Authors: Mitchell Jennifer , Siboo Ian R. , Takamatsu Daisuke , Chambers Henry F. , Sullam Paul M.
Published: May 2007

PblA and PblB are prophage‐encoded proteins of Streptococcus mitis strain SF100 that mediate binding to human platelets. The mechanism for surface expression of these proteins has been unknown, as they do not contain signal sequences or cell wall sorting motifs. We therefore assessed whether expression of these proteins was linked the lytic cycle of the prophage. Deletion of either the holin or lysin gene resulted in retention of PblA and PblB in the cytoplasm, and loss of these proteins from the cell wall. Flow cytometric analysis revealed that induction of phage replication in SF100 produced a subpopulation of cells with increased permeability. This effect was abrogated by disruption of the holin and lysin genes. Treatment of these mutants with exogenous PblA and PblB restored surface expression, apparently via binding of the proteins to cell wall choline. Loss of PblA and PblB expression was associated with decreased platelet binding in vitro, and reduced virulence in an animal model of endocarditis. Thus, expression of PblA and PblB occurs via a novel mechanism, whereby phage induction increases bacterial permeability and release of the proteins, followed by their binding to surface of viable cells. This mechanism may be important for endovascular infection.
Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress
Authors: Van Den Bogaart Geert , Hermans Nicolaas , Krasnikov Victor , Poolman Bert
Published: May 2007

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo‐bleaching, pulsed‐FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed‐FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 μm2 s−1, whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D∼0.4 μm2 s−1). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.
The shape of galaxy cluster dark matter haloes: systematics of its imprint on cluster gas and comparison to observations
Authors: Flores Ricardo A. , Allgood Brandon , Kravtsov Andrey V. , Primack Joel R. , Buote David A. , Bullock James S.
Published: May 2007

We study predictions for galaxy cluster observables that can test the statistics of dark matter halo shapes expected in a flat Λ cold dark matter (CDM) universe. We present a simple analytical model for the prediction of cluster-scale X-ray observations, approximating clusters as isothermal systems in hydrostatic equilibrium, and dark matter haloes as ellipsoids with uniform axial ratios (homeoidal ellipsoids). We test the model against high-resolution, hydrodynamic cluster simulations to gauge its reliability. We find that this simple prescription does a good job of predicting cluster X-ray ellipticities compared to the simulations as long as one focuses on cluster regions that are less sensitive to recent mergers. Based on this simple model, the distribution of cluster-size halo shapes expected in the concordance ΛCDM cosmology implies an X-ray ellipticity distribution with a mean 〈εX〉= 0.32 ± 0.01, and a scatter σε= 0.14 ± 0.01 for the mass range (1–4) × 1014h−1 M⊙. We find it important to include the mass dependence of halo shape when making comparisons to observational samples. We analyse the systematics of four observational samples of cluster ellipticities and find that our results are statistically compatible with these observations. In particular, we find remarkably good agreement between two recent ROSAT samples and ΛCDM predictions that do not include gas cooling. We also test how well our analytical model can predict Sunyaev–Zel'dovich decrement maps and find that it is less successful although still useful; the model does not perform as well as a function of flux level in this case because of the changing triaxiality of dark matter haloes as a function of radial distance. Both this effect and the changing alignment of isodensity shells of dark matter haloes leave an imprint on cluster gas that appears to be seen in observational data. Thus, dark matter haloes cannot be accurately characterized as homeoidal ellipsoids for all comparisons.
Mechanism of CO2 acquisition in an acid‐tolerant Chlamydomonas
Authors: BALKOS KONSTANTINE D. , COLMAN BRIAN
Published: May 2007

The acid‐tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using 14C‐benzoic acid (BA) or [2‐14C]‐5,5‐dimethyloxazolidine‐2,4‐dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0–7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO2 uptake and O2 evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO2 uptake. The CO2 compensation concentration of the cells ranged from 9.4 µM at pH 4.5 to 16.2 µM at pH 7.0. An examination of the kinetics of ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the Km (CO2) was 16.3 µM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO2, and the expression of external CA at neutral pH values would maintain an equilibrium CO2 concentration at the cell surface. This species does not possess a CO2‐concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low Km (CO2) Rubisco of the alga.
The stomatal response to evaporative demand persists at night in Ricinus communis plants with high nocturnal conductance
Authors: BARBOUR MARGARET M. , BUCKLEY THOMAS N.
Published: May 2007

Evidence is building that stomatal conductance to water vapour (gs) can be quite high in the dark in some species. However, it is unclear whether nocturnal opening reflects a mechanistic limitation (i.e. an inability to close at night) or an adaptive response (i.e. promoting water loss for reasons unrelated to carbon gain). Further, it is unclear if stomatal responses to leaf‐air vapour pressure difference (D) persist in the dark. We investigated nocturnal stomatal behaviour in castor bean (Ricinus communis L.) by measuring gas exchange and stomatal responses to D in the light and in the dark. Results were compared among eight growth environments [two levels for each of three treatment variables: air saturation deficit (Da), light and water availability]. In most plants, stomata remained open and sensitive to D at night. gs was typically lower at night than in the day, whereas leaf osmotic pressure (Π) was higher at night. In well‐watered plants grown at low Da, stomata were less sensitive to D in the dark than in the light, but the reverse was found for plants grown at high Da. Stomata of droughted plants were less sensitive to D in the dark than in the light regardless of growth Da. Drought also reduced gs and elevated Π in both the light and the dark, but had variable effects on stomatal sensitivity to D. These results are interpreted with the aid of models of stomatal conductance.
Seasonal variations in nitrate reductase activity and internal N pools in intertidal brown algae are correlated with ambient nitrate concentrations
Authors: YOUNG ERICA B. , DRING MATTHEW J. , SAVIDGE GRAHAM , BIRKETT DARYL A. , BERGES JOHN A.
Published: May 2007

Nitrogen metabolism was examined in the intertidal seaweeds Fucus vesiculosus, Fucus serratus, Fucus spiralis and Laminaria digitata in a temperate Irish sea lough. Internal NO3‐ storage, total N content and nitrate reductase activity (NRA) were most affected by ambient NO3‐, with highest values in winter, when ambient NO3‐ was maximum, and declined with NO3‐ during summer. In all species, NRA was six times higher in winter than in summer, and was markedly higher in Fucus species (e.g. 256 ± 33 nmol NO3‐ min−1 g−1 in F. vesiculosus versus 55 ± 17 nmol NO3‐ min−1 g−1 in L. digitata). Temperature and light were less important factors for N metabolism, but influenced in situ photosynthesis and respiration rates. NO3‐ assimilating capacity (calculated from NRA) exceeded N demand (calculated from net photosynthesis rates and C : N ratios) by a factor of 0.7–50.0, yet seaweeds stored significant NO3‐ (up to 40–86 µmol g−1). C : N ratio also increased with height in the intertidal zone (lowest in L. digitata and highest in F. spiralis), indicating that tidal emersion also significantly constrained N metabolism. These results suggest that, in contrast to the tight relationship between N and C metabolism in many microalgae, N and C metabolism could be uncoupled in marine macroalgae, which might be an important adaptation to the intertidal environment.
Potential and limits of acylcyclohexanediones for the control of blossom blight in apple and pear caused by Erwinia amylovora
Authors: Spinelli F. , Vanneste J. L. , Ciampolini F. , Cresti M. , Rademacher W. , Geider K. , Costa G.
Published: May 2007

Growth‐regulating acylcyclohexanediones such as prohexadione‐calcium and trinexapac‐ethyl have been shown to be effective in controlling fire blight infections on shoots. Since blossoms represent the primary site of infection for the fire blight pathogen, Erwinia amylovora, trinexapac‐ethyl and prohexadione‐calcium were evaluated for their ability to reduce fire blight infection on apple and pear flowers. Field experiments and experiments under controlled conditions were conducted on apple flowers for 4 years. A reduction of up to 50% of blossom blight was observed in treated plants. In addition, treatment with trinexapac‐ethyl reduced up to the 77% the percentage of fireblight‐affected flowers from which disease progressed into shoots. On pear, numbers of flower infections were reduced by a quarter and flower infections leading to diseased shoots was reduced by up to 50%. Mechanisms underlying diseased reduction following treatment with the two acylcyclohexanediones was studied using a confocal laser scanning microscope combined with a gpf‐labelled strain of E. amylovora. These non‐invasive techniques demonstrated bacterial migration was reduced by up to 60 and 66% in apple and pear xylem, respectively.
Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection
Authors: Lane C. R. , Hobden E. , Walker L. , Barton V. C. , Inman A. J. , Hughes K. J. D. , Swan H. , Colyer A. , Barker I.
Published: May 2007

Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on‐site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real‐time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number (n = 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20‐30% for P. ramorum and P. kernoviae. This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.
Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations
Authors: Abu Ahmad Y. , Girard J.‐C. , Fernandez E. , Pauquet J. , Lockhart B. E. L. , Letourmy P. , Rott P.
Published: May 2007

Two sugarcane cultivars (R570 and SP71‐6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf‐free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane‐growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71‐6163 were infected by SCYLV genotypes BRA‐PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71‐6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.
Histamine in Rheumatoid Arthritis
Authors: Adlesic M. , Verdrengh M. , Bokarewa M. , Dahlberg L. , Foster S. J. , Tarkowski A.
Published: May 2007

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a persistent inflammation of the synovium, leading to the erosion of articular cartilage and bone. Synovial mast cells and their effector molecule, histamine, receive increased attention as mediators of joint inflammation. The aim of our study was to analyse levels of free histamine in serum and joint fluid of RA patients and to evaluate the potential inflammatogenic properties of histamine in vivo and in vitro. Histamine levels were measured by an ELISA in synovial fluid and sera of RA patients and of healthy controls. Histamine levels were also assessed in plasma of RA patients undergoing anti‐TNF‐α treatment. In the murine part of the study, histamine was injected intra‐articularly in the knee joint of mice and the joints were subsequently analysed with respect to induction of inflammation. RA patients displayed significantly lower levels of histamine in circulation (0.93 ± 0.16 ng/ml) compared with the healthy controls (1.89 ± 0.45 ng/ml, P < 0.001). Locally, in synovial fluid the levels of histamine were even lower (0.37 ± 0.16 ng/ml, P < 0.0006). Long‐term anti‐TNF‐α treatment significantly increased circulating levels of histamine in RA patients. Our experiments on animals show that histamine on its own neither induces inflammation in the joint cavity nor influences the course of HMGB1 and peptidoglycan‐induced joint inflammation. Based on our experimental and clinical studies we suggest that histamine lacks harmful properties in RA.
Patterns of In vitro Lymphoproliferative Responses Among HTLV‐1‐infected Subjects: Upregulation by HTLV‐1 During HIV‐1 Co‐infection
Authors: Olah I. , Fukumori L. M. I. , Montanheiro P. , Vergara M. P. , Smid J. , Duarte A. J. S. , Penalva de Oliveira A. C. , Casseb J.
Published: May 2007

The present study evaluated the in vitro response to different mitogens and a candidin antigen (CMA) in Human T‐cell lymphotropic virus type 1 (HTLV‐1) and co‐infected HIV‐1/HTLV‐1 patients, to identify if this co‐infection may modify the spontaneous lymph proliferative response. Peripheral blood mononuclear cells from 72 healthy seronegative controls, 75 asymptomatic HTLV‐1‐infected carriers, 42 HAM/TSP cases, 33 solely HIV‐1‐infected subjects and 24 HIV‐1/HTLV‐1 patients were assayed in the presence and absence of mitogens (PHA, PWM and OKT3) and CMA. The HAM/TSP group had the highest proliferation rate at 3 and 6 days after culture. HAM/TSP cases showed decreased response to PHA, compared with asymptomatic HTLV‐1 subjects, and most important, the co‐infected HIV‐1/HTLV‐1 cases presented a similar response to HTLV‐1‐infected subjects after 3 days of culture. The singles HIV‐1‐infected group had decreased in vitro response. It appears that during co‐infection, the HTLV‐1 regulatory proteins overwhelm the action of HIV‐1 regulatory proteins.
SHA1, a novel RING finger protein, functions in shoot apical meristem maintenance in Arabidopsis
Authors: Sonoda Yutaka , Yao Shan‐Guo , Sako Kaori , Sato Takeo , Kato Wataru , Ohto Masa‐aki , Ichikawa Takanari , Matsui Minami , Yamaguchi Junji , Ikeda Akira
Published: May 2007

Post‐embryonic plant growth is dependent on a functional shoot apical meristem (SAM) that provides cells for continuous development of new aerial organs. However, how the SAM is dynamically maintained during vegetative development remains largely unclear. We report here the characterization of a new SAM maintenance mutant, sha1‐1 (shoot apical meristem arrest 1‐1), that shows a primary SAM‐deficient phenotype at the adult stage. The SHA1 gene encodes a novel RING finger protein, and is expressed most intensely in the shoot apex. We show that, in the sha1‐1 mutant, the primary SAM develops normally during the juvenile vegetative stage, but cell layer structure becomes disorganized after entering the adult vegetative stage, resulting in a dysfunctional SAM that cannot initiate floral primordia. The sha1‐1 SAM terminates completely at the stage when the wild‐type begins to bolt, producing adult plants with a primary inflorescence‐deficient phenotype. These observations indicate that SHA1, a putative E3 ligase, is required for post‐embryonic SAM maintenance by controlling proper cellular organization.
Self‐incompatibility of Prunus tenella and evidence that reproductively isolated species of Prunus have different SFB alleles coupled with an identical S‐RNase allele
Authors: Šurbanovski Nada , Tobutt Kenneth R. , Konstantinović Miroslav , Maksimović Vesna , Sargent Daniel J. , Stevanović Vladimir , Ortega Encarnación , Bošković Radovan I.
Published: May 2007

Many species of Prunus display an S‐RNase‐based gametophytic self‐incompatibility (SI), controlled by a single highly polymorphic multigene complex termed the S‐locus. This comprises tightly linked stylar‐ and pollen‐expressed genes that determine the specificity of the SI response. We investigated SI of Prunus tenella, a wild species found in small, isolated populations on the Balkan peninsula, initially by pollination experiments and identifying stylar‐expressed RNase alleles. Nine P. tenella S‐RNase alleles (S1–S9) were cloned; their sequence analysis showed a very high ratio of non‐synonymous to synonymous nucleotide substitutions (Ka/Ks) and revealed that S‐RNase alleles of P. tenella, unlike those of Prunus dulcis, show positive selection in all regions except the conserved regions and that between C2 and RHV. Remarkably, S8‐RNase, was found to be identical to S1‐RNase from Prunus avium, a species that does not interbreed with P. tenella and, except for just one amino acid, to S11 of P. dulcis. However, the corresponding introns and S‐RNase–SFB intergenic regions showed considerable differences. Moreover, protein sequences of the pollen‐expressed SFB alleles were not identical, harbouring 12 amino‐acid replacements between those of P. tenella SFB8 and P. avium SFB1. Implications of this finding for hypotheses about the evolution of new S‐specificities are discussed.
Light‐regulated large‐scale reorganization of chromatin during the floral transition in Arabidopsis
Authors: Tessadori Federico , Schulkes Roeland Kees , Driel Roel van , Fransz Paul
Published: May 2007

The floral transition marks the switch from vegetative to reproductive growth, and is controlled by different pathways responsive to endogenous and exogenous cues. The developmental switch is accompanied by local changes in chromatin such as histone modifications. In this study we demonstrate large‐scale reorganization of chromatin in rosette leaves during the floral transition. An extensive reduction in chromocenters prior to bolting is followed by a recovery of the heterochromatin domains after elongation of the floral stem. The transient reduction in chromocenters is a result of relocation away from chromocenters of methylated DNA sequences, 5S rDNA and interspersed pericentromeric repeats, but not of 45S rDNA or the 180‐bp centromere tandem repeats. Moreover, fluorescence in situ hybridization analysis revealed decondensation of chromatin in gene‐rich regions. A mutant analysis indicated that the blue‐light photoreceptor CRYPTOCHROME 2 is involved in triggering chromatin decondensation, suggesting a light‐signaling pathway towards large‐scale chromatin modulation.
The Arabidopsis ABA‐deficient mutant aba4 demonstrates that the major route for stress‐induced ABA accumulation is via neoxanthin isomers
Authors: North Helen M. , Almeida Aurélie De , Boutin Jean‐Pierre , Frey Anne , To Alexandra , Botran Lucy , Sotta Bruno , Marion‐Poll Annie
Published: May 2007

A novel abscisic acid (ABA)‐deficient mutant, aba4, was identified in a screen for paclobutrazol‐resistant germination. Compared with wild‐type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map‐based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans‐neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA‐deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9‐cis‐epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.
The KNOX gene SHOOT MERISTEMLESS is required for the development of reproductive meristematic tissues in Arabidopsis
Authors: Scofield Simon , Dewitte Walter , Murray James A.H.
Published: May 2007

In Arabidopsis, the central stem cells of the shoot apical meristem (SAM) are indefinitely maintained, whereas those in floral meristems differentiate into female gametophyte‐bearing organs termed carpels. The class 1 KNOX genes encode homeodomain transcription factors that function variously in the establishment and maintenance of the SAM, and have also been implicated in carpel development. Here we show that the KNOX gene SHOOT MERISTEMLESS (STM) induces carpel formation and promotes homeotic conversion of ovules to carpels when ectopically expressed in flowers, as previously reported for the related gene KNAT2. In contrast to KNAT2, loss of which confers no phenotype, we show using inducible RNA interference and mutational analysis that progressive loss of STM causes floral phenotypes ranging from reduced formation of placental tissues and inhibited carpel fusion to complete loss of carpel development. These effects result neither from failure to establish the central stem cell niche nor from reduced floral homeotic gene expression, but rather indicate a specific requirement for STM in carpel initiation, as further supported by the loss of leafy carpelloid features in stm leafy double mutants. Activation of carpel development by STM is independent of LEAFY and WUSCHEL, but requires the function of AGAMOUS. The essential role for STM in carpel development, together with its previous reported role in the SAM, shows that, despite the existence of several partially redundant paralogous genes, STM provides the critical KNOX function in the development of both vegetative and reproductive meristematic tissues.
Arabidopsis Ovate Family Protein 1 is a transcriptional repressor that suppresses cell elongation
Authors: Wang Shucai , Chang Ying , Guo Jianjun , Chen Jin‐Gui
Published: May 2007

Transcription factors regulate multiple aspects of plant growth and development. Here we report the identification and functional analysis of a plant‐specific, novel transcription factor in Arabidopsis. We isolated a dominant, gain‐of‐function mutant that displays reduced lengths in all aerial organs including hypocotyl, rosette leaf, cauline leaf, inflorescence stem, floral organs and silique. Molecular cloning revealed that these phenotypes are caused by elevated expression of the Arabidopsis thaliana Ovate Family Protein 1 (AtOFP1). This mutant was designated as Atofp1‐1D. We show that the altered morphology of Atofp1‐1D mutant is caused by reduced cell length resulting from reduced cell elongation, and demonstrate that a mutant harboring a transposon insertion that disrupts the OVATE domain of AtOFP1 is indistinguishable from wild‐type plants. Plants overexpressing other closely related AtOFP genes phenocopy plants overexpressing AtOFP1, implying a possible overlapping function among members of the AtOFP gene family. We found that AtOFP1 localizes in the nucleus, and that AtOFP1 functions as an active transcriptional repressor. Chromatin immunoprecipitation results indicated that AtGA20ox1, a gene encoding the key enzyme in GA biosynthesis, is a target gene regulated by AtOFP1. Consistent with this, exogenous gibberellic acid can partially restore defects in cell elongation in plants overexpressing AtOFP1, suggesting that such a reduced cell elongation is caused, in part, by the deficiency in gibberellin biosynthesis. Taken together, our results indicate that AtOFP1 is an active transcriptional repressor that has a role in regulating cell elongation in plants.
Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth
Authors: Kong Hongzhi , Landherr Lena L. , Frohlich Michael W. , Leebens‐Mack Jim , Ma Hong , DePamphilis Claude W.
Published: May 2007

Gene duplication plays important roles in organismal evolution, because duplicate genes provide raw materials for the evolution of mechanisms controlling physiological and/or morphological novelties. Gene duplication can occur via several mechanisms, including segmental duplication, tandem duplication and retroposition. Although segmental and tandem duplications have been found to be important for the expansion of a number of multigene families, the contribution of retroposition is not clear. Here we show that plant SKP1 genes have evolved by multiple duplication events from a single ancestral copy in the most recent common ancestor (MRCA) of eudicots and monocots, resulting in 19 ASK (Arabidopsis SKP1‐like) and 28 OSK (Oryza SKP1‐like) genes. The estimated birth rates are more than ten times the average rate of gene duplication, and are even higher than that of other rapidly duplicating plant genes, such as type I MADS box genes, R genes, and genes encoding receptor‐like kinases. Further analyses suggest that a relatively large proportion of the duplication events may be explained by tandem duplication, but few, if any, are likely to be due to segmental duplication. In addition, by mapping the gain/loss of a specific intron on gene phylogenies, and by searching for the features that characterize retrogenes/retrosequences, we show that retroposition is an important mechanism for expansion of the plant SKP1 gene family. Specifically, we propose that two and three ancient retroposition events occurred in lineages leading to Arabidopsis and rice, respectively, followed by repeated tandem duplications and chromosome rearrangements. Our study represents a thorough investigation showing that retroposition can play an important role in the evolution of a plant gene family whose members do not encode mobile elements.
Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold‐regulated gene expression with modifications in metabolite content
Authors: Kaplan Fatma , Kopka Joachim , Sung Dong Yul , Zhao Wei , Popp Mick , Porat Ron , Guy Charles L.
Published: May 2007

Exposure of Arabidopsis to low temperatures results in cold acclimation where freezing tolerance is enhanced. To achieve a wider view of the role of transcriptome to biochemical changes that occur during cold acclimation, analyses of concurrent transcript and metabolite changes during cold acclimation was performed revealing the dynamics of selected gene–metabolite relationships. Exposure to low temperature resulted in broad transcriptional and metabolite responses. Principal component analysis revealed sequentially progressive, global changes in both gene expression and metabolite profiles during cold acclimation. Changes in transcript abundance for many metabolic processes, including protein amino acid biosynthetic pathways and soluble carbohydrates, during cold acclimation were observed. For some metabolic processes, changes in transcript abundance temporally correlated with changes in metabolite levels. For other metabolic processes, changes in transcript levels were not correlated with changes in metabolite levels. The present findings demonstrate that regulatory processes independent of transcript abundance represent a key part of the metabolic adjustments that occur during cold acclimation.
Identification of a flavin‐monooxygenase as the S‐oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis
Authors: Hansen Bjarne G. , Kliebenstein Daniel J. , Halkier Barbara A.
Published: May 2007

The cancer‐preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4‐methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer‐preventive properties, whereas the isothiocyanate of 4‐methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin‐monooxygenase (FMO) enzyme, FMOGS‐OX1, which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs. This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMOGS‐OX1 overexpression lines and an FMOGS‐OX1 knock‐out mutant of Arabidopsis. The FMOGS‐OX1 overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4‐methylsulfinylbutyl GSL in seeds. Identification of FMOGS‐OX1 provides a molecular tool for breeding of Brassica vegetable crops with increased levels of this important GSL, which has implications for production of functional foods enriched with the cancer‐preventive sulforaphane.
Microcolinearity and genome evolution in theAdhAregion of diploid and polyploid cotton (Gossypium)
Authors: Grover Corrinne E. , Kim HyeRan , Wing Rod A. , Paterson Andrew H. , Wendel Jonathan F.
Published: May 2007

Genome sizes vary by several orders of magnitude, driven by mechanisms such as illegitimate recombination and transposable element proliferation. Prior analysis of theCesAregion in two cotton genomes that diverged 5–10 million years ago (Ma), and acquired a twofold difference in genome size, revealed extensive local conservation of genic and intergenic regions, with no evidence of the global genome size difference. The present study extends the comparison to include BAC sequences surrounding the gene encoding alcohol dehydrogenase A (AdhA) from four cotton genomes: the two co‐resident genomes (ATand DT) of the allotetraploid,Gossypium hirsutum, as well as the model diploid progenitors,Gossypium arboreum(A) andGossypium raimondii(D). In contrast to earlier work, evolution in theAdhAregion reflects, in a microcosm, the overall difference in genome size, with a nearly twofold difference in aligned sequence length. Most size differences may be attributed to differential accumulation of retroelements during divergence of the genome diploids from their common ancestor, but in addition there has been a biased accumulation of small deletions, such that those in the smaller D genome are on average twice as large as those in the larger A genome. The data also provide evidence for the global phenomenon of ‘genomic downsizing’ in polyploids shortly after formation. This in part reflects a higher frequency of small deletions post‐polyploidization, and increased illegitimate recombination. In conjunction with previous work, the data here confirm the conclusion that genome size evolution reflects many forces that collectively operate heterogeneously among genomic regions.
Purification of the M flax‐rust resistance protein expressed in Pichia pastoris
Authors: Schmidt Simon A. , Williams Simon J. , Wang Ching‐I A. , Sornaraj Pradeep , James Ben , Kobe Bostjan , Dodds Peter N. , Ellis Jeffrey G. , Anderson Peter A.
Published: May 2007

The M flax‐rust resistance (R) gene is predicted to encode a 150‐kDa protein of the Toll‐interleukin‐like receptor‐nucleotide binding site‐leucine rich repeat (TIR‐NBS‐LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N‐terminally 9× His‐tagged protein, in which the first 21 amino acids of the predicted wild‐type protein are deleted. Expression was achieved using a high cell density fed‐batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole‐cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.
ABA‐Hypersensitive Germination1 encodes a protein phosphatase 2C, an essential component of abscisic acid signaling in Arabidopsis seed
Authors: Nishimura Noriyuki , Yoshida Tomo , Kitahata Nobutaka , Asami Tadao , Shinozaki Kazuo , Hirayama Takashi
Published: May 2007

The phytohormone abscisic acid (ABA) regulates physiologically important stress and developmental responses in plants. To reveal the mechanism of response to ABA, we isolated several novel ABA‐hypersensitive Arabidopsis thaliana mutants, named ahg (ABA‐hypersensitive germination). ahg1‐1 mutants showed hypersensitivity to ABA, NaCl, KCl, mannitol, glucose and sucrose during germination and post‐germination growth, but did not display any significant phenotypes in adult plants. ahg1‐1 seeds accumulated slightly more ABA before stratification and showed increased seed dormancy. Map‐based cloning of AHG1 revealed that ahg1‐1 has a nonsense mutation in a gene encoding a novel protein phosphatase 2C (PP2C). We previously showed that the ahg3‐1 mutant has a point mutation in the AtPP2CA gene, which encodes another PP2C that has a major role in the ABA response in seeds (Yoshida et al., 2006b). The levels of AHG1 mRNA were higher in dry seeds and increased during late seed maturation – an expression pattern similar to that of ABI5. Transcriptome analysis revealed that, in ABA‐treated germinating seeds, many seed‐specific genes and ABA‐inducible genes were highly expressed in ahg1‐1 and ahg3‐1 mutants compared with the wild‐type. Detailed analysis suggested differences between the functions of AHG1 and AHG3. Dozens of genes were expressed more strongly in the ahg1‐1 mutant than in ahg3‐1. Promoter–GUS analyses demonstrated both overlapping and distinct expression patterns in seed. In addition, the ahg1‐1 ahg3‐1 double mutant was more hypersensitive than either monogenic mutant. These results suggest that AHG1 has specific functions in seed development and germination, shared partly with AHG3.
Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways
Authors: Stefanovic Aleksandra , Ribot Cécile , Rouached Hatem , Wang Yong , Chong Julie , Belbahri Lassaad , Delessert Syndie , Poirier Yves
Published: May 2007

The PHO1 family comprises 11 members in Arabidopsis thaliana. In order to decipher the role of these genes in inorganic phosphate (Pi) transport and homeostasis, complementation of the pho1 mutant, deficient in loading Pi to the root xylem, was determined by the expression of the PHO1 homologous genes under the control of the PHO1 promoter. Only PHO1 and the homologue PHO1;H1 could complement pho1. The PHO1;H1 promoter was active in the vascular cylinder of roots and shoots. Expression of PHO1;H1 was very low in Pi‐sufficient plants, but was strongly induced under Pi‐deficient conditions. T‐DNA knock‐out mutants of PHO1;H1 neither showed growth defects nor alteration in Pi transport dynamics, or Pi content, compared with wild type. However, the double mutant pho1/pho1;h1 showed a strong reduction in growth and in the capacity to transfer Pi from the root to the shoot compared with pho1. Grafting experiments revealed that phenotypes associated with the pho1 and pho1/pho1;h1 mutants were linked to the lack of gene expression in the root. The increased expression of PHO1;H1 under Pi deficiency was largely controlled by the transcription factor PHR1 and was suppressed by the phosphate analogue phosphite, whereas the increase of PHO1 expression was independent of PHR1 and was not influenced by phosphite. Together, these data reveal that although transfer of Pi to the root xylem vessel is primarily mediated by PHO1, the homologue PHO1;H1 also contributes to Pi loading to the xylem, and that the two corresponding genes are regulated by Pi deficiency by distinct signal transduction pathways.
Immunogenic tagging of chloroplasts allows their isolation from defined cell types
Authors: Truernit Elisabeth , Hibberd Julian M.
Published: May 2007

Chloroplast structure varies depending on cell type. Currently it is difficult to obtain insight into how differences in chloroplast structure relate to function, as it is often not possible to isolate chloroplasts from specific cells. To address this, we have developed an approach that involves labelling chloroplasts from individual cell types by placing the foreign yellow fluorescent protein (YFP) on their outer surface, and then isolating those labelled chloroplasts immunogenically. Cell specificity is achieved through the use of enhancer trap lines. When whole leaves are homogenized, a mixture of labelled and unlabelled chloroplasts is released, but magnetic beads coated with antibodies to the green fluorescent protein (GFP) allow the labelled chloroplasts to be isolated. Chloroplasts from spongy mesophyll, vascular and epidermal cells of Arabidopsis thaliana were obtained in this way, and semi‐quantitative RT‐PCR showed that the abundance of various chloroplast transcripts differed between these three cell types. The approach is based on genetic logic, and so could be applied to the isolation of various organelles or subcellular compartments from transformable organisms other than A. thaliana.
Deficiency of mitochondrial fumarase activity in tomato plants impairs photosynthesis via an effect on stomatal function
Authors: Nunes‐Nesi Adriano , Carrari Fernando , Gibon Yves , Sulpice Ronan , Lytovchenko Anna , Fisahn Joachim , Graham James , Ratcliffe R. George , Sweetlove Lee J. , Fernie Alisdair R.
Published: May 2007

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of a fumarate hydratase (fumarase) gene in the antisense orientation and exhibiting considerable reductions in the mitochondrial activity of this enzyme show impaired photosynthesis. The rate of the tricarboxylic acid cycle was reduced in the transformants relative to the other major pathways of carbohydrate oxidation and the plants were characterized by a restricted rate of dark respiration. However, biochemical analyses revealed relatively little alteration in leaf metabolism as a consequence of reducing the fumarase activity. That said, in comparison to wild‐type plants, CO2 assimilation was reduced by up to 50% under atmospheric conditions and plants were characterized by a reduced biomass on a whole plant basis. Analysis of further photosynthetic parameters revealed that there was little difference in pigment content in the transformants but that the rate of transpiration and stomatal conductance was markedly reduced. Analysis of the response of the rate of photosynthesis to variation in the concentration of CO2 confirmed that this restriction was due to a deficiency in stomatal function.