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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.

 

FERONIA (FER) receptor kinase plays versatile roles in plant growth and development, biotic and abiotic stress responses, and reproduction. Autophagy is a conserved cellular recycling process that is critical for balancing plant growth and stress responses. Target of Rapamycin (TOR) has been shown to be a master regulator of autophagy. Our previous multi-omics analysis with loss-of-function fer-4 mutant implicated that FER functions in the autophagy pathway. We further demonstrated here that the fer-4 mutant displayed constitutive autophagy, and FER is required for TOR kinase activity measured by S6K1 phosphorylation and by root growth inhibition assay to TOR kinase inhibitor AZD8055. Taken together, our study provides a previously unknown mechanism by which FER functions through TOR to negatively regulate autophagy. 

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.

 

The receptor kinase FERONIA (FER) is a versatile regulator of plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new FER functions, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of Arabidopsis (Arabidopsis thaliana) wild-type and fer-4 loss-of-function mutant plants. Gene ontology terms for phytohormone signaling, abiotic stress, and biotic stress were significantly enriched among differentially expressed transcripts, differentially abundant proteins, and/or misphosphorylated proteins, in agreement with the known roles for FER in these processes. Analysis of multiomics data and subsequent experimental evidence revealed previously unknown functions for FER in endoplasmic reticulum (ER) body formation and glucosinolate biosynthesis. FER functions through the transcription factor NAI1 to mediate ER body formation. FER also negatively regulates indole glucosinolate biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors is hypophosphorylated in the fer-4 mutant and demonstrated that FER acts through the transcription factor ABA INSENSITIVE5 (ABI5) to negatively regulate the ABA response during cotyledon greening. Our integrated omics study, therefore, reveals novel functions for FER and provides new insights into the underlying mechanisms of FER function.

 

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins. 

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.