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Abstract 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. 

Abstract The G‐protein complex is a cytoplasmic on–off molecular switch that is set by plasma membrane receptors that activate upon binding of its cognate extracellular agonist. In animals, the default setting is the “off” resting state, while in plants, the default state is constitutively “on” but repressed by a plasma membrane receptor‐like protein. De‐repression appears to involve specific phosphorylation of key elements of the G‐protein complex and possibly target proteins that are positioned downstream of this complex. To address this possibility, protein abundance and phosphorylation state are quantified in wild type and G‐protein deficient Arabidopsis roots in the unstimulated resting state. A total of 3246 phosphorylated and 8141 non‐modified protein groups are identified. It has been found that 428 phosphorylation sites decrease and 509 sites increase in abundance in the G‐protein quadrupole mutant lacking an operable G‐protein‐complex. Kinases with known roles in G‐protein signaling including MAP KINASE 6 and FERONIA are differentially phosphorylated along with many other proteins now implicated in the control of G‐protein signaling. Taken together, these datasets will enable the discovery of novel proteins and biological processes dependent on G‐protein signaling. 

The Mla (Mildew resistance locus a) of barley (Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich repeat (CC-NLR) receptor often require the HRS complex (HSP90, RAR1, and SGT1) to function. However, functional analysis of Sgt1 has been particularly difficult, as deletions are often lethal. Recently, we identified rar3 (required for Mla6 resistance 3), an in-frame Sgt1 ΔKL308-309 mutation in the SGT1-specific domain, that alters resistance conferred by MLA but without lethality. Here, we use autoactive MLA6 and recombinant yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90 to determine that this mutation weakens but does not entirely disrupt the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1 ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022. 

Recent advances in single-cell proteomics for animal systems could be adapted for plants to increase our understanding of plant development, response to stimuli, and cell-to-cell signaling.