| Somatic embryogenesis (SE) is the developmental reprogramming of somaticcells toward the embryogenesis pathway and is a notable illustration of cell totipotency.SE is a unique developmental pathway and has been viewed as a potential model systemfor the study of the basic mechanisms of development reprogramming among the highereukaryotic organism. Study on SE could provide a useful way to understand plantdevelopment such as embryogenesis and may have merit as a study presenting anapplication in plant biology due to its biological theory and economical importance. Ourmain results could be summarized as the following points:1. We conducted a systematic assessment and comparative study on the biochemicaland cytogenetics characteristics of cultured cotton cells during the process of somaticembryogenesis. All staged cultures were widely investigated in this assay. Efficient plantregeneration system from cell cultures can be established in cotton (2n=52) bymonitoring ploidy level and visual selection of the cultures. Cell and tissue ectogenesismanipulation combined with flow cytometry was employed to study the whole process ofdedifferentiation and redifferentiation. We identified two phases of chromatindecondensation during the dedifferentiation and redifferentiation that undediestotipotency, reprogramming, and formation of new stem cell lineages. At the same time,sharp increase in the ratio of IAA, iPs at the same stage of cell dedifferentiation andredifferentiation process serve as distinct biochemical maker of dedifferentiation andsomatic embryogenesis initiation with the unique feature. Our results suggest the twophases of chromatin decondensation, associated with endogenous hormone dynamicactivity of cultured cotton cell, underlies dedifferentiation and redifferentiationreprogramming during the entire somatic embryogenesis process in cotton.2. Transcriptome profiling and clustering of SE-associated transcript tags during thewhole SE process in cotton was comprehensively analyzed.To identify genes involved inSE, subtractive polymerase chain reaction (PCR) was performed to generate transcriptshighly enriched for SE-related genes, using cDNA prepared from a mixture ofembryogenic callus and preglobular somatic embryos, as the tester, and cDNA from nonembryogenic callus, as the driver. After differential screening and subsequentconfirmation by reverse Northern blot analysis, a total of 671 differentially expressedcDNA fragments were identified, and 242 unigenes significantly up-regulated duringcotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCRanalysis of representative cases, including most previously published SE-related genes inplants. In total, more than half had not been identified previously as SE-related genes,including dominant crucial genes involved in transcription, posttranscription, andtransportation, and about one-third had not been reported previously to GenBank or wereexpected to be unknown, or newly identified genes. We used cDNA arrays to furtherinvestigate the expression patterns of these genes in differentiating gradient cultures,ranging from proembryogenic masses to somatic embryos at every stage. Our resultssuggested that a concerted mechanism involving multiple cellular pathways isresponsible for cotton SE. This report represents a systematic and comprehensiveanalysis of genes involved in the process of somatic embryogenesis.3. Characterization and comparative analyses of transcriptomes during somaticembrygenesis and zygote embryogenesis in cotton were conducted systemically. Ourresults suggest that there are common gene expression profiling and regulation modebetween these two similar biological processes, while there are also some differentcharacteristic on biological process, cellular component,molecular function andmetabolic pathways. We dissected and identified all staged zygote and endosperm duringtheir development in cotton ovule using microscope. Fertilization-induced genesspecially expressed in zygote cell and genes preferentially expressed during zygote andendosperm development were isolated and identified in cotton.4. We propose an economical-yet effective-computational method for genome-widediscovery of genes responsive to auxin in A. thaliana. Evaluation of our result bycomparing it with published microarray data and literature showed that this method hasan accuracy rate of 65.2% (309/474) of predicting candidate genes responsive to auxin.We investigated experimental functional information and chromosome distribution mapsin the Arabidopsis genome of the candidate auxin-responsive genes predicted.Bioinformatic data mining for candidate genes with Gene Ontology and KEGG metabolic pathways associated with these putative auxin-responsive genes were alsoidentified. We believe the results can significantly reduce the number of identifiedpotential auxin target genes and also provide functional clues for annotating parts ofgenes that lack functional information.5. To better understand the concerted mechanism involving multiple cellularpathways and the relationships between molecular events during this importantreprogramming process, we used bioinformatics and pathway database to construct adraft molecular interaction network inferred from transcriptionally regulated SE-relatedgenes, from functional genomics assays readout to high level biological datainterpretation. Here, logical SE association networks predict various cellular pathwaysand genes involved in SE, which are validated in cultured cells by independentlaboratories. A complex molecular system was unraveled by SE association network. Byanalyzing the draft network, the processes of cell death and cell proliferation wererevealed to be significant components of SE as the extremely noteworthy SE associationbiomarkers. In addition, the cross talks and dynamic balance in the interactions among allhormones (Auxin, Ethylene, Brassinolide, Gibberellins, Cytochalasin and ABA) whichregulate (positive or negative) the distinct processes of cell death and cell proliferationsuggest they are pivotal in switching cell fate during the developmental plasticity of SEthrough coordinated interactions with many developmental signaling pathways. Thisreport represents a comprehensive analysis of the SE association network in plants.Combining transcriptionally regulated genes with logical association networks facilitatesunderstanding of the functional genomics of complex molecular systems. The molecularinteraction network draft based on high-throughput interaction datasets from modelorganism will provide a rich framework for SE research.6. Robustness functional analysis of key genes, using series of experiments ofoverexpression and RNA interference (RNAi) strategies, were performed to elucidatetheir specific roles in embryogenesis and enhance our understanding of the cell fatedetermination and polarization process in plants, as well as to testify the roles andinteraction relation of SE association biomarkers in SE association regulatory networks.Recently, our studies on functional analysis of these key regulatory factors during early embryogenesis get primary positive results. A gateway cloning vector set forhigh-throughput functional analysis of genes and proteins were used effectively in theseinvestigations.
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