Abstract
Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin, rapid changes in transcript and protein abundance occur in hypocotyls, and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin‐regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA‐BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T‐DNA allele of IRR found that it had a wild‐type (WT) hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid‐auxin‐phytochrome (BAP) regulatory module compared to WT, including transcription factors that regulate brassinosteroid, auxin, and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence data and genetic complementation analysis with SALK_015201C, we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase‐related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb‐9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin‐dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T‐DNA stocks.
Highlights
TERACTING FACTORs (PIFs), collectively referred to as the BZR/AUXIN RESPONSE FACTORs (ARFs)/PHYTOCHROME INTERACTING FACTORS (PIFs) module or the ‘BAP module’ (Bouré et al., In plants, phytohormones work in concert to coordinate 2019)
We recently characterized auxin responsive proteomes irr-2 to confirm this single T deletion within the PHYB locus. (Kelley et al, 2017) and used these data to perform a. This analysis confirmed the presence of a single base pair reverse genetic screen (Page and Grossniklaus, 2002) for deletion in PHYB in the SALK_015201 background that was seedlings with altered auxin-dependent phenotypes
One of not observed in Col-0 or irr-2 (Supplemental Figure 4). This the candidate genes included in this screen was AT3G23900 single nucleotide deletion in the PHYB coding sequence is which has been recently characterized as IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), a novel RNA
Summary
Part of the Cell and Developmental Biology Commons, Genetics Commons, and the Plant Sciences Commons. The complete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ gdcb_las_pubs/265. For information on how to cite this item, please visit http://lib.dr.iastate.edu/howtocite.html. Slim shady is a novel allele of PHYTOCHROME B present in the T-DNA line SALK_015201
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