Cell Biology & Molecular Genetics

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    THE PHYTOHORMONE ETHYLENE: (I) INVESTIGATING THE MOLECULAR FUNCTION OF RTE1 AND (II) INSIGHTS ON THE EVOLUTION OF THE ETHYLENE BIOSYNTHESIS AND SIGNALING PATHWAYS
    (2017) Clay, John; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ethylene is an important phytohormone that regulates growth, development and stress responses in land plants and charophycean green algae. In Arabidopsis thaliana, ethylene is perceived by a family of five receptors. One of these five receptors, ETR1, is dependent on REVERSION-TO-ETHYLENE1 (RTE1) and Cytochrome B5 (Cb5) while the other four receptors are not. We found that RTE1 and Cb5 interact in planta and used genetic analyses to place Cb5 upstream of RTE1 in the ethylene signaling pathway. After comparing different ethylene receptors we identified an N-terminally localized proline that is important in determining whether a receptor is RTE1-dependent. Our results suggest that Cb5 receives electrons from upstream redox molecules, passes these electrons to RTE1; RTE1 is then able to activate the ETR1 receptor possibly by acting a molecular chaperone that refolds the ETR1 receptor into an active conformation. The ethylene signal transduction pathway is functionally conserved in the charophycean green algae such as Spirogyra pratensis, suggesting that this signaling pathway was present in the common ancestor of charophytes and land plants over 450 million years ago. However, it is unclear whether the central regulator of ethylene response, EIN2, was conserved in charophytes. Furthermore, the details of ethylene biosynthesis in charophytes were unresolved. After examining the genomes and transcriptomes of many green algae we are able to report that EIN2 is conserved in most charophytes and even some of the more distantly related chlorophycean green algae. Moreover, the Spirogyra EIN2 is functionally conserved and able to activate ethylene responses in Arabidopsis. Ethylene is synthesized via a two-step reaction involving the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase (ACS), followed by oxidation of ACC to ethylene gas by the enzyme ACC oxidase (ACO). We identified S. pratensis ACS homologs and demonstrated that S. pratensis can synthesize ACC. S. pratensis lacks ACO homologs but we find it is still capable of producing low levels of ethylene. From our results we conclude that the ethylene biosynthesis and signaling pathways were established in early charophytes allowing these algae to establish ethylene as an important signalling molecule.
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    Uncovering the evolutionary origin of plant molecular processes: comparison of Coleochaete (Coleochaetales) and Spirogyra (Zygnematales) transcriptomes
    (2010-05-25) Timme, Ruth E; Delwiche, Charles F
    Background: The large and diverse land plant lineage is nested within a clade of fresh water green algae, the charophytes. Collection of genome-scale data for land plants and other organisms over the past decade has invigorated the field of evolutionary biology. One of the core questions in the field asks: how did a colonization event by a green algae over 450 mya lead to one of the most successful lineages on the tree of life? This question can best be answered using the comparative method, the first step of which is to gather genome-scale data across closely related lineages to land plants. Before sequencing an entire genome it is useful to first gather transcriptome data: it is less expensive, it targets the protein coding regions of the genome, and provides support for gene models for future genome sequencing. We built Expressed Sequence Tag (EST) libraries for two charophyte species, Coleochaete orbicularis (Coleochaetales) and Spirogyra pratensis (Zygnematales). We used both Sanger sequencing and next generation 454 sequencing to cover as much of the transcriptome as possible. Results: Our sequencing effort for Spirogyra pratensis yielded 9,984 5' Sanger reads plus 598,460 GS FLX Standard 454 sequences; Coleochaete orbicularis yielded 4,992 5' Sanger reads plus 673,811 GS FLX Titanium 454 sequences. After clustering S. pratensis yielded 12,000 unique transcripts, or unigenes, and C. orbicularis yielded 19,000. Both transcriptomes were very plant-like, i.e. most of the transcripts were more similar to streptophytes (land plants + charophyte green algae) than to other green algae in the sister group chlorophytes. BLAST results of several land plant genes hypothesized to be important in early land plant evolution resulted in high quality hits in both transcriptomes revealing putative orthologs ripe for follow-up studies. Conclusions: Two main conclusions were drawn from this study. One illustrates the utility of next generation sequencing for transcriptome studies: larger scale data collection at a lower cost enabled us to cover a considerable portion of the transcriptome for both species. And, two, that the charophyte green algal transcriptoms are remarkably plant-like, which gives them the unique capacity to be major players for future evolutionary genomic studies addressing origin of land plant questions.