MORF2-mediated plastidial retrograde signaling is involved in stress response and skotomorphogenesis beyond RNA editing.

Retrograde signaling modulates the expression of nuclear genome-encoded organelle proteins to adjust organelle function in response to environmental cues. MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) was initially recognized as a plastidial RNA-editing factor but recently shown to interact with GUN1. Given the central role of GUN1 in chloroplast retrograde signaling and the unviable phenotype of morf2 mutants that is inconsistent with many viable mutants involved in RNA editing, we hypothesized that MORF2 has functions either dosage dependent or beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we were able to reduce the MORF2 transcripts in a controlled manner. In addition to MORF2-dosage dependent RNA-editing errors, we discovered that reducing MORF2 by iCRISPRi stimulated the expression of stress responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased accumulation of hydrogen peroxide. These findings along with previous discoveries suggest that MORF2 is an effective regulator involved in plastidial metabolic pathways whose reduction can readily activate multiple retrograde signaling molecules possibly involving reactive oxygen species to adjust plant growth. In addition, our newly developed iCRISPRi approach provided a novel genetic tool for quantitative reverse genetics studies on hub genes in plants.

A Machine Learning Approach to Prioritizing Functionally Active F-box Members in Arabidopsis thaliana.

Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana. Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization.

Generation of a fertile ask1 mutant uncovers a comprehensive set of SCF-mediated intracellular functions.

Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi-subunit Skp1-Cullin1-F-box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F-box proteins. Although both sequence divergence and polymorphism of F-box genes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F-box and Cullin1 in the complex, we systematically analyzed the functional influence of a well-characterized Arabidopsis Skp1-Like1 (ASK1) Ds insertion allele, ask1, in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia-0 (Col-0), we partially rescued the fertility of this otherwise sterile ask1 allele in Landsberg erecta, thus providing experimental evidence showing the polymorphic roles of SCF complexes. This ask1 mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col-0. RNA-Seq-based transcriptome analysis of ask1 uncovered a large spectrum of SCF functions, which is greater than a 10-fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far-red light/phyA-mediated photomorphogenesis. Such diverse roles are consistent with the 20-30% reduction of ubiquitylation events in ask1 estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertile ask1 mutant allowed us to uncover a comprehensive set of SCF functions.