The role of RNA decay in the abiotic stress responses of Brachypodium
Date
2018
Authors
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Publisher
University of Delaware
Abstract
Brachypodium distachyon (Brachypodium) is a small annual grass species related to cereal grains such as wheat, barley, rice, and maize as well as bioenergy crops such as switchgrass and sorghum. Since the proposal of Brachypodium as a model for the grasses over 500 Bdi-miRNAs have been annotated in miRBase. While a significant number of miRNAs have been identified which are highly conserved across plants, little research has been done with respect to the conservation of miRNA targets. Plant responses to abiotic stresses are regulated by diverse pathways many of which involve miRNAs; however, it can be difficult to identify miRNA guided gene regulation when the miRNA is not the primary regulator of the target mRNA. ☐ To investigate miRNA target conservation and stress response involvement, a set of PARE (Parallel Analysis of RNA Ends) libraries totaling over 2 billion reads was constructed and sequenced from Brachypodium, switchgrass, and sorghum representing the first public release of degradome data from the latter two species. Analysis of these data provided not only PARE evidence for miRNA guided cleavage of over 7,000 predicted target mRNAs in Brachypodium, but also evidence for miRNA guided cleavage of over 1,000 homologous transcripts in sorghum and switchgrass. A pipeline was constructed to compare RNA-seq and PARE data made from Brachypodium plants exposed to various abiotic stress conditions. This resulted in the identification of 44 miRNA targets which exhibit stress regulated cleavage. Time course experiments were performed to reveal the relationship between miR393ab, miR169a, miR394ab, and their respective targets throughout the first 36 hours of cold stress. These experiments resulted in the first evidence that miR169a and miR394ab are associated with the cold stress response of Brachypodium. ☐ Post-transcriptional gene regulation is not limited to miRNA related mechanisms. Multiple instances of regulation by means of RNA stability have been observed in plants. While our previous pipeline focused on miRNAs, we developed a new type of PARE analysis with the aim of identifying any instances of post-transcriptional gene regulation. The use of this pipeline to analyze PARE libraries made from cold treated plants resulted in the identification of a set of 1,399 transcripts. A GO (Gene Ontology) term enrichment analysis provided evidence for a number of these transcripts having biological significance to the cold stress response in Brachypodium. To measure changes in mRNA stability on a global scale, we used cordycepin, an adenosine analogue, to inhibit transcription in plants exposed to cold and heat stresses. We sampled plant tissue 30, 60, and 120 minutes after exposure to cordycepin and constructed RNA-seq libraries from these samples. We estimated mRNA decay rates for individual transcripts by fitting the normalized transcript abundances, as reported by RNA-seq, to an exponential regression model. We found a total of 35 transcripts with half-lives under 60 minutes during control conditions which were stabilized under cold stress conditions. Additionally, exposure to heat resulted in a decrease in the mRNA half-lives of 82 transcripts to below 60 minutes, while causing an increase to above 60 minutes for 11 transcripts. These data represent the first genomic analysis of mRNA half-lives in Brachypodium. ☐ We were interested in generating a bdxrn4 mutant due to loss of XRN4 function in Arabidopsis resulting in a stabilization of 5'-monophosphorylated decay intermediates, such as those from miRNA guided cleavage events, as well as a potential association with the stress responses. We constructed RNAi vectors with hairpins targeting two different regions of the BdXRN4 transcript; however, multiple transformation attempts resulted in plantlets dying shortly after shoot formation. A shift in strategy to the CRISPR-Cas9 mediated method for genome editing resulting in the generation of 24 successfully transformed T0 plants which exhibited a short bushy phenotype. Only 11 of those plants produced seed; however, we were able to use Sanger sequencing to identify mutant alleles present in the T2 generation of three lines. Of those, the mutations present in two lines appeared stable and allowed for the identification of two plants homozygous for two different frameshift inducing alleles. Interestingly, despite these two mutant lines originating from independent transformation events, both of the frameshift inducing alleles contained a single nucleotide insertion at the same location with the only difference between the alleles being the identity of the inserted base. These mutants exhibited a sickly phenotype that was much more pronounced than those observed in Arabidopsis atxrn4. Plants homozygous for either of these frameshift inducing alleles displayed a significantly reduced growth rate, low germination rate, delayed flowering, loss of apical dominance, and a physically short and bushy phenotype characterized by an increased number of tillers. To gain a better understanding of the changes in gene regulation which could explain these phenotypes, we constructed RNA-seq libraries from both of these mutants and two wild type control plants. Analysis of this data using edgeR allowed for the identification of over 1,000 differentially regulated genes. These included two genes, MOTHEROF FT AND TFL1 b (BdMFT1b) and FLOWERING LOCUS T LIKE (BdFTL), the dysregulation of which could explain the lowered germination rate and delayed flowering respectively. These plants represent the first XRN4 loss of function mutant in any monocot species and will facilitate studies that lead to a deeper understanding of RNA decay in Brachypodium and other monocots.
Description
Keywords
Bioenergy, Bioinformatics, Brachypodium, miRNA, Post-transcriptional, RNA