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A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of tasiRNAs (TAS1–4) and microRNAs (pri-MIR163, pri-MIR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2–10 amino acids), and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.

 

Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies have revealed connections between these two pathways, but a system-wide view of their interplay is still missing. • We quantified the level of 23,975 transcripts, 11,183 proteins, and 27,887phosphorylation sites in wild-type Arabidopsis thalianaand inmutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (B IN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively.• We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity.• Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR- or TORC-regulated growth and autophagy. 

Brassinosteroids (BRs) are plant steroid hormones that regulate cell division and stress response. Here we use a systems biology approach to integrate multi-omic datasets and unravel the molecular signaling events of BR response inArabidopsis. We profile the levels of 26,669 transcripts, 9,533 protein groups, and 26,617 phosphorylation sites fromArabidopsisseedlings treated with brassinolide (BL) for six different lengths of time. We then construct a network inference pipeline called Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) to integrate these data. We use our network predictions to identify putative phosphorylation sites on BES1 and experimentally validate their importance. Additionally, we identify BRONTOSAURUS (BRON) as a transcription factor that regulates cell division, and we show thatBRONexpression is modulated by BR-responsive kinases and transcription factors. This work demonstrates the power of integrative network analysis applied to multi-omic data and provides fundamental insights into the molecular signaling events occurring during BR response.

 

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 calledslim shady, in an annotated insertion line inIMMUNOREGULATORY RNA‐BINDING PROTEIN(IRR). Overexpression of theIRRgene failed to rescue theslim shadyphenotype and characterization of a second T‐DNA allele of IRR found that it had a wild‐type (WT) hypocotyl length. Theslim shadymutant 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 shadyseedlings 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 inPHYTOCHROME Bwas responsible for theslim shadyphenotype. 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 withphyb‐9confirmed thatslim shadyis a mutant allele ofPHYB. 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.