Zhihua Hua

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next‐generation sequencing (NGS) approaches, such as rRNA‐depleted long non‐coding (lnc) RNA‐seq or targeted amplicon‐seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, I developed a rapid and cost‐effective long‐read sequencing approach, termed Target‐Indexed‐PCR (TIP) sequencing, for digital quantification of RNA editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high‐fidelity PCR amplification with Oxford Nanopore sequencing and custom in‐house Perl and Python scripts for streamlined data processing, including barcode‐based demultiplexing, strand reorientation, alignment to a pseudo‐genome, manual editing‐site inspection, and splicing variant identification. As a proof of concept, TIP sequencing was applied to ndhB and ndhD transcripts, two chloroplast NAD ( P ) H dehydrogenase genes with the highest number of known editing sites in Arabidopsis thaliana . These transcripts were analyzed both in an inducible CRISPR interference (iCRISPRi) system targeting MORF2 , a key RNA‐editing factor, and in MORF2‐YFP transgenic lines with either overexpression or co‐suppression silencing. My findings revealed dose‐ and development‐dependent impacts of MORF2 on C‐to‐U editing efficiency. Moreover, I identified an accumulation of intron‐retaining ndhB transcripts, specifically in Dex‐treated iCRISPRi lines and in both MORF2‐YFP overexpression and silencing rosette leaves, indicating impaired chloroplast splicing functions when MORF2 expression is perturbed beyond an as‐yet‐undefined threshold. The platform achieves single‐molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA‐seq. Collectively, this study establishes TIP sequencing as a versatile, scalable, and affordable tool for targeted post‐transcriptional analysis in plant organelles and expands our understanding of MORF2 's role in chloroplast RNA maturation. By overcoming key limitations of existing approaches, TIP sequencing enables routine, site‐specific quantification of post‐transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle–nucleus communication.

Nearly all eukaryotic proteins are turned over by the ubiquitin (Ub)-26S proteasome system (UPS). Despite its broad cellular roles, only a handful of UPS members, particularly the Ub E3 ligases that specifically recognize a protein for ubiquitylation, have been characterized in plants to date. The challenge arises from the transient recognition and rapid degradation of ubiquitylation substrates by the UPS. To tackle this challenge, the emerging biotinylation-based proximity labeling (PL) offers an exciting tool for enriching transient interactors of Ub E3 ligases. In this study, we examined the efficacy of TurboID in identifying substrates of Arabidopsis Skp1-cullin1-F-box (SCF) ligases. We demonstrate that the Arabidopsis Skp1 Like (ASK)1-TurboID is not fully functioning in planta, which led us to discover a novel antagonism between biotinylation and ubiquitylation in regulating protein stability in vivo. This discovery lowers the effectiveness of PL in ubiquitylome studies. However, using one long-known SCF substrate, phytochrome A, we succeeded to apply its TurboID fusion for complementing the far-red-light response of the phyA-211 null mutant allele, suggesting an efficacy of PL in characterizing single ubiquitylation pathways. This study highlighted a limitation of PL in ubiquitylome studies, discovered a new antagonistic pathway of biotinylation, and developed a theoretical guidance for future PL-based characterization of ubiquitylation pathways.

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, we developed a rapid and cost-effective long-read sequencing approach, termed premium PCR sequencing, for digital quantification of RNA-editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification and comparison. Using this platform, we analyzed the ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites, in an inducible CRISPR interference (iCRISPRi) system targeting MORF2 , a key RNA-editing factor. Our results revealed MORF2 dosage-dependent reductions in C-to-U editing efficiency, with significant defects observed in the strongly repressed P1-12 line. Moreover, we identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines, indicating impaired chloroplast splicing functions upon MORF2 suppression. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq, making it broadly accessible. This study establishes premium PCR sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2’s role in chloroplast RNA maturation. Significance Statement: We present a rapid, affordable, and reproducible method for digital quantification of RNA editing and intron retention in plant organellar transcripts using nanopore-based long-read sequencing. This platform overcomes key limitations of existing approaches and enables routine, site-specific analysis of post-transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle–nucleus communication.

In plants, the ubiquitin (Ub)-26S proteasome system (UPS) regulates numerous biological functions by selectively targeting proteins for ubiquitylation and degradation. However, the regulation of Ub itself on plant growth and development remains unclear. To demonstrate a possible impact of Ub supply, as seen in animals and flies, we carefully analyzed the growth and developmental phenotypes of two different poly-Ub (UBQ) gene overexpression plants of Arabidopsis thaliana. One is transformed with hexa-6His-UBQ (designated 6HU), driven by the cauliflower mosaic virus 35S promoter, while the other expresses hexa-6His-TEV-UBQ (designated 6HTU), driven by the endogenous promoter of UBQ10. We discovered that 6HU and 6HTU had contrasting seed yields. Compared to wildtype (WT), the former exhibited a reduced seed yield, while the latter showed an increased seed production that was attributed to enhanced growth vigor and an elevated silique number per plant. However, reduced seed sizes were common in both 6HU and 6HTU. Differences in the activity and size of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with WT, suggestive of a contributory role of UBQ expression in proteasome assembly and function. Collectively, our findings demonstrated that exogenous expression of recombinant Ub may optimize plant growth and development by influencing the UPS activities via structural variance, expression patterns, and abundance of free Ub supply.

Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivaling several other major systems, such as transcription regulation and epigenetics, I described the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarized the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I proposed a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I presented new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of Ub E3 ligase genes.

In the crowded and confined space of a cell, numerous proteins work collaboratively in various subsystems, such as metabolic pathways, organelle compartments, and complexes, to regulate cell growth and development

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.

The ubiquitin-26S proteasome system and autophagy are two major protein degradation machineries encoded in all eukaryotic organisms. While the UPS is responsible for the turnover of short-lived and/or soluble misfolded proteins under normal growth conditions, the autophagy-lysosomal/vacuolar protein degradation machinery is activated under stress conditions to remove long-lived proteins in the forms of aggregates, either soluble or insoluble, in the cytoplasm and damaged organelles. Recent discoveries suggested an integrative function of these two seemly independent systems for maintaining the proteome homeostasis. One such integration is represented by their reciprocal degradation, in which the small 76-amino acid peptide, ubiquitin, plays an important role as the central signaling hub. In this review, we summarized the current knowledge about the activity control of proteasome and autophagosome at their structural organization, biophysical states, and turnover levels from yeast and mammals to plants. Through comprehensive literature studies, we presented puzzling questions that are awaiting to be solved and proposed exciting new research directions that may shed light on the molecular mechanisms underlying the biological function of protein degradation.

SUMMARY: Functional studies of the ubiquitin–26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant's life involve UPS‐mediated turnover of abnormal or short‐lived proteins. However, the role of the UPS during development, including in seeds and fruits, remains to be determined in detail, although mutants of several of its core elements are known to be embryonically lethal. Unfortunately, early termination of embryogenesis limits the possibility to characterize the activities of the UPS in reproductive organs. Given both the economic and the societal impact of reproductive production, such studies are indispensable. Here, we systematically compared expression of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings, developing siliques, and embryos of Arabidopsis thaliana. Since autophagy plays the second largest role in maintaining proteome stability, we parallelly studied three rate‐limiting enzymes that are involved in autophagy flux. Our experiments unexpectedly discovered that, in contrast to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques or embryos toward maturation while the autophagy flux rises despite the nutrient‐rich condition. We also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy flux is suppressed in seedlings by UPS‐mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques, which in turn stimulates a bulk autophagic degradation of proteasomes. Collectively, our study of the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early silique and/or seed development, highlighting their interactions during development.