Multimodal Imaging of Cotton Wool Spots in Branch Retinal Vein Occlusion.

PURPOSE: To describe and follow cotton wool spots (CWS) in branch retinal vein occlusion (BRVO) using multimodal imaging. METHODS: In this prospective cohort study including 24 patients with new-onset BRVO, CWS were described and analyzed in color fundus photography (CF), spectral domain optical coherence tomography (SD-OCT), infrared (IR) and fluorescein angiography (FA) every 3 months for 3 years. The CWS area on SD-OCT and CF was evaluated using OCT-Tool-Kit software: CWS were marked in each single OCT B-scan and the software calculated the area by interpolation. RESULTS: 29 central CWS lesions were found. 100% of these CWS were visible on SD-OCT, 100% on FA and 86.2% on IR imaging, but only 65.5% on CF imaging. CWS were visible for 12.4 ± 7.5 months on SD-OCT, for 4.4 ± 3 months and 4.3 ± 3.4 months on CF and on IR, respectively, and for 17.5 ± 7.1 months on FA. The evaluated CWS area on SD-OCT was larger than on CF (0.26 ± 0.17 mm(2) vs. 0.13 ± 0.1 mm(2), p < 0.0001). The CWS area on SD-OCT and surrounding pathology such as intraretinal cysts, avascular zones and intraretinal hemorrhage were predictive for how long CWS remained visible (r(2) = 0.497, p < 0.002). CONCLUSIONS: The lifetime and presentation of CWS in BRVO seem comparable to other diseases. SD-OCT shows a higher sensitivity for detecting CWS compared to CF. The duration of visibility of CWS varies among different image modalities and depends on the surrounding pathology and the CWS size.

A non-persistently transmitted-virus induces a pull?push strategy inits aphid vector to optimize transmission and spread

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly,generally adapting to each type of virus?vector relationship in a way that enhances transmissionefficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant,cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypiiGlover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferousaphids changed their alighting, settling and probing behaviour activities over time when exposed toCMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate fromCMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), butshowed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min afterrelease. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected overmock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage andaphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph(EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of shortsuperficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (sec-ond hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much lesstime spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour includingan early increase in the number of short superficial probes and intracellular punctures followed by aphloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in aNP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting,settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spreadof the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NPmanner.

Polyploid formation created unique avenues for response to selection in Gossypium (cotton)

A detailed restriction fragment length polymorphism map was used to determine the chromosomal locations and subgenomic distributions of quantitative trait loci (QTLs) segregating in a cross between cultivars of allotetraploid (AADD) Gossypium hirsutum (“Upland” cotton) and Gossypium barbadense (“Sea Island,” “Pima,” or “Egyptian” cotton) that differ markedly in the quality and quantity of seed epidermal fibers. Most QTLs influencing fiber quality and yield are located on the “D” subgenome, derived from an ancestor that does not produce spinnable fibers. D subgenome QTLs may partly account for the fact that domestication and breeding of tetraploid cottons has resulted in fiber yield and quality levels superior to those achieved by parallel improvement of “A” genome diploid cottons. The merger of two genomes with different evolutionary histories in a common nucleus appears to offer unique avenues for phenotypic response to selection. This may partly compensate for reduction in quantitative variation associated with polyploid formation and be one basis for the prominence of polyploids among extant angiosperms. These findings impel molecular dissection of the roles of divergent subgenomes in quantitative inheritance in many other polyploids and further exploration of both “synthetic” polyploids and exotic diploid genotypes for agriculturally useful variation.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

(E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ ≈ 150 μM; Km for Mn2+ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km ≈ 0.6 μM; Vrel = 100) and Mg2+ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 μM; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus)

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 × 105 cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase–PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

Duplicated genes evolve independently after polyploid formation in cotton

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF– and the derivative mutant–microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.

The Involvement of Hydrogen Peroxide in the Differentiation of Secondary Walls in Cotton Fibers1

H2O2 is a widespread molecule in many biological systems. It is created enzymatically in living cells during various oxidation reactions and by leakage of electrons from the electron transport chains. Depending on the concentration H2O2 can induce cell protective responses, programmed cell death, or necrosis. Here we provide evidence that H2O2 may function as a developmental signal in the differentiation of secondary walls in cotton (Gossypium hirsutum) fibers. Three lines of evidence support this conclusion: (a) the period of H2O2 generation coincided with the onset of secondary wall deposition, (b) inhibition of H2O2 production or scavenging the available H2O2 from the system prevented the wall differentiation process, and (c) exogenous addition of H2O2 prematurely promoted secondary wall formation in young fibers. Furthermore, we provide support for the concept that H2O2 generation could be mediated by the expression of the small GTPase Rac, the accumulation of which was shown previously to be strongly induced during the onset of secondary wall differentiation. In support of Rac's role in the activation of NADPH oxidase and the generation of reactive oxygen species, we transformed soybean (Glycine max) and Arabidopsis cells with mutated Rac genes. Transformation with a dominantly activated cotton Rac13 gene resulted in constitutively higher levels of H2O2, whereas transformation with the antisense and especially with dominant-negative Rac constructs decreased the levels of H2O2.

GTPase Activity and Biochemical Characterization of a Recombinant Cotton Fiber Annexin1

A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An “add-back experiment” was performed to study the effect of the recombinant annexin on β-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.

A Fiberless Seed Mutation in Cotton Is Associated with Lack of Fiber Cell Initiation in Ovule Epidermis and Alterations in Sucrose Synthase Expression and Carbon Partitioning in Developing Seeds1

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

Genes Involved in Osmoregulation during Turgor-Driven Cell Expansion of Developing Cotton Fibers Are Differentially Regulated1

Cotton (Gossypium hirsutum L.) fibers are single-celled trichomes that synchronously undergo a phase of rapid cell expansion, then a phase including secondary cell wall deposition, and finally maturation. To determine if there is coordinated regulation of gene expression during fiber expansion, we analyzed the expression of components involved in turgor regulation and a cytoskeletal protein by measuring levels of mRNA and protein accumulation and enzyme activity. Fragments of the genes for the plasma membrane proton-translocating ATPase, vacuole-ATPase, proton-translocating pyrophosphatase (PPase), phosphoenolpyruvate carboxylase, major intrinsic protein, and α-tubulin were amplified by polymerase chain reaction and used as probes in ribonuclease protection assays of RNA from a fiber developmental series, revealing two discrete patterns of mRNA accumulation. Transcripts of all but the PPase accumulated to highest levels during the period of peak expansion (+12–15 d postanthesis [dpa]), then declined with the onset of secondary cell wall synthesis. The PPase was constitutively expressed through fiber development. Activity of the two proton-translocating-ATPases peaked at +15 dpa, whereas PPase activity peaked at +20 dpa, suggesting that all are involved in the process of cell expansion but with varying roles. Patterns of protein accumulation and enzyme activity for some of the proteins examined suggest posttranslational regulation through fiber development.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.