Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR. Citrus ovules were harvested from the excised ovaries of different flower developmental stages. These developing stages were decided on the basis of morphological and histological characteristics of flower and female gametophyte, respectively. We selected, Pre anthesis, post anthesis, and initial fruit set stage of flower to excise ovules from Polyembryonic cultivar Vaniglia Sanguigno and Monoembryonic cultivar Temple. Leaf tissue of Vaniglia Sanguigno was used to check the overall transcriptome level differences between leaf and ovule tissues. For each sample, three technical replicates were used.
Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR.
Project description:Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 30,171 sets expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. Young, healthy Valencia sweet orange (C. sinensis) plants were graft inoculated with budwood from Ca. L. asiaticus-infected citrus plants. Prior to the innocualtion, the plants were confirmed to be Ca. L. asiaticus-free in ordinary and quantitative PCR tests. The presence of the bacteria in the inoculated plants was confirmed in both conventional and quantitative PCR with specific primers to Ca. L. asiaticus. The stem and root samples used for RNA extraction and hybridization on Affymetrix microarrays were obtained from three symptomatic and three healthy control trees of similar size, approximately 1 year after inoculation.
Project description:Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium Candidatus Liberibacter spp. In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 30,171 sets expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories.
Project description:Pathogens can trigger a broad array of changes in gene expression in plants. In this study we report the changes in gene expression patterns that occurred when greenhouse grown Washington Navel oranges (C. sinensis) was graft innoculated with citrus pathogens. Candidatus Liberibacter asiaticus, Spiroplasma citri, and two isolates of citrus tristeza virus were studied.
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Citrus sinensis tissues (including leaves, flowers and fruit). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features, such as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and fruit of Citrus sinensis. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and flowers and the Guanidinium-free for fruits, and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Erik Mirkov for providing the plant material, as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Background: MicroRNAs play important roles in the adaptive responses of plants to nutrient deficiencies. Here, we sequenced two small RNA libraries from B-deficient and -sufficient (control) Citrus sinensis leaves, respectively, using Illumina sequencing in order to identify the potential miRNAs related to the tolerance of citrus to B-deficiency. Results: Ninety one (83 known and 8 novel) up- and 81 (75 known and 6 novel) downregulated miRNAs were isolated from B-deficient leaves. The great alteration of miRNA expression might contribute to the tolerance of citrus to B-deficiency. The adaptive responses of miRNAs to B-deficiency might related to several aspects: (a) attenuation of plant growth and development by repressing auxin signaling due to decreased TIR1 level and ARF-mediated gene expression by altering the expression of miR393, miR160 and miR3946; (b) maintaining leaf phenotype and enhancing the stress tolerance by up-regulating NACs targeted by miR159, miR782, miR3946 and miR7539; (c) activation of the stress responses and antioxidant system through down-regulating the expression of miR164, miR6260, miR5929, miR6214, miR3946 and miR3446; (d) decreasing the expression of major facilitator superfamily protein genes targeted by miR5037, thus lowering B export from plants. Also, B-deficiency-induced downregulation of miR408 might play a role in plant tolerance to B-deficiency by regulating Cu homeostasis and enhancing superoxide dismutase activity. Conclusions: Our study reveals some novel responses of citrus to B-deficiency, which increase our understanding of the adaptive mechanisms of citrus to B-deficiency at the miRNA (post-transcriptional) level.
Project description:<p>Traumatic brain injury is a significant cause of mortality in young adults and disability in all age groups. Secondary injury involving various processes such as oxidative stress, which induced neuronal apoptosis, ensued afterward. This study aimed to analyze the active compounds of Citrus sinensis and predict its antioxidative activity in traumatic brain injury. Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS) was used to identify compounds in ethanol extract of Citrus sinensis peel. We analyzed pharmacokinetics, blood-brain barrier (BBB) permeability and toxicity of active compounds in Citrus sinensis peel using SwissADME and OSIRIS. The compounds have good blood-brain barrier permeability conducted for molecular docking study to identify molecular interaction with Keap1 (Kelch-like ECH Associated Protein 1) and NMDA (N-methyl-D-aspartate) proteins using PyRx, PyMol and Discovery Studio software. Results of the LC-HRMS examination obtained 16 active compounds contained in the ethanol extract of the orange peel. Nootkatone, alminoprofen, linoleic acid, chanoclavine, scoparone and tangeretin are predicted to pass the blood-brain barrier. Active compounds of Citrus sinensis strongly bond against Keap1 and NMDA, especially Scoparone and Nootkatone. The strong binding affinity of scoparone-Keap1 was -5.0, more than the control ligand, and nootkatone-NMDA was -7.8, similar to the control ligand. Active compounds of Citrus sinensis peel showed inhibitory potentials, good pharmacokinetics and toxicity profiles against Keap1 and NMDA. These findings suggested that ethanol extract of Citrus sinensis peels has the potential as an oxidative stress inhibitor for brain injury therapy.</p>
Project description:Candidatus Liberibacter asiaticus infection of citrus is characterized by symptom variability within and among organs. In order to identify molecular processes involved in the regulation of organ response to Ca. Liberibacter infection, the gene expression patterns in C. sinensis leaf, stem, and root was examined in Affymetrix microarray. Our analyses showed that Ca. L. asiaticus reprograms several cellular and metabolic processes in C. sinensis, with most categories regulated in leaves, followed by stems and least in roots. Among them, we identified genes whose expression is regulated in organ-specific manner, reflecting organ specialization in the molecular response to Ca. L. asiaticus. Differences in gene expression were expected between these organs because of functional divergence among them. Two-year old Valencia sweet orange (C. sinensis) plants were graft inoculated with budwood from Ca. L. asiaticus-infected citrus plants. Successful infection of the inoculated plants was confirmed in both conventional and quantitative PCR with specific primers to Ca. L. asiaticus. The stem and root samples used for RNA extraction and hybridization on Affymetrix microarrays were obtained from three symptomatic and three healthy control trees of similar size, 16 months after the inoculation.