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 Mimulus guttatus tissues (including leaves, flowers and roots). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features 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 roots of Mimulus guttatus, strain âIM62â. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), 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 John Willis for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:About one third of all angiosperm species produce flowers with petals fused into a corolla tube. As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii. Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPRESSOR OF GENE SILENCING 3 cause a dramatic decrease in tasiARF abundance and a moderate up-regulation of Auxin Response Factor 3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. Integrating our molecular and phenotypic analyses of the tasiRNA-ARF pathway in Mimulus with historical insights from morphological and anatomical studies in various sympetalous species, we propose a new conceptual model for the developmental genetic control of corolla tube formation.
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 Mimulus guttatus tissues (including leaves, flowers and roots). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features 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.
Project description:The seeds of angiosperms are comprised of three main tissues--the seed coat, embryo, and endosperm--whose coordinated development will enable the next generation to disperse with sufficient maternal resources and germinate when conditions are favorable. We use high-throughput RNA sequencing technology to characterize the developmental dynamics of gene expression in whole seeds resulting from a compatible cross between two species of the Mimulus guttatus complex.We find that development in ovules and seeds involves the activation of the majority of annotated genes in M. guttatus (64-67%), and the differential expression over time of 6,691 genes, including 424 transcription factors. Most of the genes we detected (69%) were expressed at all stages, from ovules to heart-stage embryo seeds. We also detected and validated four genes exhibiting paternally-biased expression (MDB13, ATXR5, DnaJ and BGAL11). Intriguingly, three of our validated PEGs are have imprinted homologues in other plant species, suggesting that these proteins perform a shared role in endosperm development among distantly related plant taxa. Additionally, the overlap in gene expression profiles between stages of development suggests that most genes fulfill multiple developmental and biological roles. Future analyses should examine whether different regions of the seed (embryo, endosperm, and seed coat) have unique patterns of gene expression, and the extent to which spatial coordination and regulation of gene expression may play a role in regulating seed development.
Project description:The action of natural selection across heterogeneous natural landscapes drives local adaptation and the formation of plant ecotypes, the precursors to new species. Plant ecotypes typically differ greatly in morphology, physiology, and development, yet we have a poor understanding of the underlying genetic basis of this divergence. Despite their importance, studies of the molecular underpinnings of ecotypic divergence through developmental stages are rare. In this study, we compared gene expression at different developmental time points between two locally adapted populations derived from the coastal perennial and inland annual ecotypes of the yellow monkeyflower, Mimulus guttatus. We conducted gene co-expression network analyses to identify putative genetic and molecular mechanisms driving ecotypic divergence and evaluated gene ontology enrichment. Overall, we found significant global expression differences between the ecotypes and among developmental stages. We also found evidence to support the hypothesis that interacting hormone pathways, especially the gibberellin and jasmonic acid pathways, may play a key role in annual versus perennial ecotype divergence. Further, genes within known chromosomal inversions were more likely to be differentially expressed than genes in colinear regions, but only in leaves and not for floral buds. Collectively, our results reinforce our existing hypotheses about how ecotype formation has occurred in this system through shifts in hormone biosynthesis/signaling and help advance our understanding of the evolution of annuality versus perenniality in plants.