slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

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.

GAUT10 is required for Arabidopsis root cell differentiation and elongation

“…Transcriptomic analyses were performed as previously described (Dash et al ., 2021). Col-0 and homozygous gaut10-3 seeds were surface sterilized using 50% bleach and 0.01% Triton X-100 for 10 minutes and then washed five times with sterile water.…”

Application of Multi-Omics Technologies to the Study of Phytochromes in Plants

“…The downstream molecules of phyB, PIF4, PIF5, and HY5 are also affected [20,80,81], where PIF4 and its co-activator HEMERA (HMR) recruit a tail subunit called MED14 to activate auxin biosynthesis and signal transduction [82]. Another study found that slim shady is a mutant allele of phyB using the brassinosteroidauxin-phytochrome (BAP) module [54] and a steroid phytohormone, brassinolide (BR), which promotes hypocotyl elongation [83], and was confirmed by RNA-seq analysis to be affected by phyB, which inhibits the transcription of BRI1-EMS-SUPPRESSOR 1 (BES1) gene and negatively regulates BR biosynthesis [53]. Finally, a plant thermal morphology with a shortened hypocotyl and reduced lateral root development is constructed, and these plants show higher heat resistance.…”

The Arabidopsis T‐DNA mutant SALK_008491 carries a 14‐kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress

“…The majority of Arabidopsis mutants studied by plant scientists are publicly available transfer-DNA (T-DNA) insertion lines. Over the last years, it has become clear that many T-DNA lines carry secondsite insertions and deletions of varying degree and sizes (Dash et al, 2021;OMalley et al, 2015). Even complex genome reorganizations have been reported (Jupe et al, 2019).…”