The biogenesis and function of plant mitochondria relies on the expression of the organellar genome, which involves extensive post-transcriptional processing of organellar transcripts in order to generate mature functional RNAs. This includes the removal of introns from many essential organellar transcripts through splicing, a process mediated by diverse nuclear-encoded RNA-processing factors, including numerous members of the pentatricopeptide repeat (PPR) protein family. Here, we report the analysis of PPR9 (At1g03560), a mitochondrial P-class PPR protein originally identified in genetic screens for essential protein cofactors. Loss of PPR9 function results in embryonic arrest at early stages of seed development. In vitro rescued homozygous ppr9 mutant plants could be recovered and maintained using a modified in vitro embryo-rescue method. The mutants display delayed growth and development which are associated with altered mitochondrial functions. Molecular analyses reveal that PPR9 functions in the splicing of several mitochondrial transcripts encoding respiratory complex I (CI) subunits, including nad2 intron 3 and nad7 introns 1 and 2. Accordingly, CI assembly is disrupted in ppr9 plants, resulting in altered OXPHOS activity and broad physiological defects. Together, these findings identify PPR9 as a key regulator of mt-RNA maturation in Arabidopsis, enabling CI biogenesis and optimal respiratory functions, and further highlight the roles of nuclear-encoded factors in coordinating mitochondria functions and (early) plant growth and development. Our study identifies PPR9 as a key mitochondrial RNA-processing factor required for the splicing of multiple group II introns that reside within genes encoding subunits essential for respiratory complex I biogenesis in Arabidopsis. By linking nuclear control of RNA maturation to mitochondrial function and plant development, this work emphasizes a critical layer of coordination between organellar gene expression, mitochondria function and developmental regulation in land plants.
The ubiquitination/deubiquitination system is a key regulatory mechanism for maintaining proteome homeostasis in eukaryotic cells. Deubiquitinating enzymes (DUBs), which remove ubiquitin from substrate proteins, regulate plant development, stress responses, and other physiological processes. Although DUBs have emerged as important regulators of plant growth and stress responses, their systematic genomic characterization in white birch (Betula platyphylla) remains limited, restricting our understanding of their potential roles in the reproduction of woody plants. In this study, 56 BpDUB genes were identified in white birch (Betula platyphylla) and classified into six conserved subfamilies (UBP, OTU, UCH, MJD, JAMM, and MINDY). Comparative and synteny analyses revealed that DUB genes are evolutionarily conserved while exhibiting lineage-specific divergence across angiosperms. Promoter analysis showed that BpDUBs harbor diverse cis-acting elements associated with growth and development, stress responses, hormone signaling, and transcription factor binding. Treatment with the broad-spectrum DUB inhibitor PR-619 markedly impaired both pollen germination and pollen tube elongation, supporting the involvement of DUB activity in pollen development. RNA-seq analysis showed that BpUBP3 exhibited stage-specific induction during pollen germination, and AS-ODN-mediated downregulation of BpUBP3 reduced pollen germination and pollen tube growth. These findings provide preliminary evidence supporting a role for BpUBP3 in pollen germination and pollen tube growth and provide a basis for further investigation of DUB functions in reproductive development in white birch. The identified DUB family is associated with pollen development in Betula platyphylla, with BpUBP3 showing potential involvement in pollen germination and pollen tube growth.
Plant derived multifunctional inhibitors (MFIs) are an important class of plant defense proteins that disrupt insect digestion by simultaneously impairing protein and carbohydrate catabolism. With the increasing knowledge of inhibitors of plant enzymes, MFIs have become a separate functional group. In this review we propose an extended definition that encompasses the classical inhibitors acting on structurally unrelated enzymes and also inhibitors acting on enzymes of the same substrate pathway, e.g. α-amylase/α-glucosidase and trypsin/chymotrypsin systems. Most of the MFIs consist of single polypeptide chains with spatially separated inhibitory sites, which allow simultaneous inhibition of several enzyme targets. The structural organization, inhibitory mechanism and evolutionary diversification of these inhibitors are reviewed with emphasis on the major families including Bowman-Birk, Kunitz-type and cereal trypsin/α-amylase inhibitors. Their domain architecture, disulfide bond patterns and structure–function relationships are discussed in view of their remarkable stability and functional versatility. Recent advances in computational structural biology, including molecular docking, molecular dynamics simulations, and AlphaFold-based protein structure prediction, have promoted mechanistic insights and enabled structure-guided engineering of improved inhibitors. The review also discusses the evolutionary origin of MFIs through gene duplication, domain shuffling, sequence diversification and adaptive evolution, and emerging approaches such as protein engineering, recombinant expression, synthetic biology and genome editing. Finally, the applications in crop protection, development of biopesticides and food biotechnology, as well as the challenges regarding insect adaptation, evolution of resistance, biosafety and ecological impacts are critically discussed. Bridging current gaps in structural characterization and in vivo validation is necessary for the translation of MFIs into sustainable agricultural and biotechnological applications. This review proposes an extended definition of plant multifunctional inhibitors, highlighting their structural diversity, evolutionary origins, and emerging applications in structure-guided crop protection and synthetic biology.
Genetically encoded reporters are indispensable tools for monitoring gene expression, transformation efficiency, and diverse cellular processes in plants. The betalain-based RUBY reporter has recently emerged as a convenient visual marker requiring no substrates or specialized equipment, and it has been widely used for a variety of applications. However, the developmental and metabolic consequences of its expression remain unexplored. Here, we show that independent transgenic lines of Arabidopsis thaliana expressing RUBY exhibit profound alterations in growth and reproductive development. RUBY-expressing plants were significantly smaller, produced fewer leaves, flowered later, and frequently developed malformed siliques with reduced seed yield. In addition, root architecture was severely impaired, with a marked reduction in the number of lateral roots. This phenotype was not rescued by indole-3-acetic acid (IAA). Targeted metabolomic profiling revealed widespread alterations in amino acid and hormone pools, including reduced indole-3-acetic acid and gibberellin, alongside accumulation of auxin precursors. Disruption of RUBY expression by suppressing the third enzyme (glucosyltransferase) restored the RUBY-associated phenotypes to wild-type. Together, our findings show that RUBY expression in Arabidopsis thaliana is not metabolically neutral; rather, it perturbs both development and metabolism by disrupting hormone and amino acid homeostasis. These results highlight reporter-associated trade-offs and underscore the necessity of regulated expression systems, such as inducible or tissue-specific expression, to expand the utility of RUBY in plant research and biotechnology. RUBY offers a convenient visual reporter of transgene expression but can disrupt hormone and amino acid homeostasis, impairing plant growth and development and highlighting the need for inducible or tissue-specific expression strategies.
The formation of nitrogen-fixing symbiotic nodules is a complex process that involves extensive transcriptional reprogramming of legume roots to enable intracellular accommodation of host compatible rhizobia. This process requires coordinated regulation of hundreds to thousands of genes, including the induction of highly expressed genes, traditionally referred to as nodulin genes. While the functions of many nodulins have been elucidated, a substantial number remained poorly characterized, or their functions are still unclear. One such gene, NOD25, was originally identified based on differential hybridization between roots and nodules and is expressed at exceptionally high levels in Medicago nodules. MtNOD25-like proteins in IRLC legumes exhibit a repetitive modular structure flanked by conserved N- and C-terminal regions, with clade-specific repeat organization. To understand the role of NOD25 in symbiotic nitrogen fixation, we generated loss-of-function mutants in Medicago truncatula using CRISPR/Cas9-mediated genome editing. Four independent Mtnod25 mutant lines displayed normal growth under symbiotic conditions and showed no detectable defects in nodule colonization, symbiosome formation, or bacteroid differentiation. These results indicate that MtNOD25 is not essential for symbiosis in M. truncatula under the conditions tested. However, the biological function of MtNOD25 has not yet been elucidated, and its highly specialized evolutionary conservation suggests that it may have a specific, non-essential role in modulating or fine-tuning symbiotic processes under particular environmental or physiological conditions that remain to be defined. Our results demonstrate that NOD25-like nodule-specific proteins possess conserved terminal domains and a rapidly evolving central region, suggesting functional diversification in IRLC legumes. Despite strong nodule-specific expression and symbiosome localization, loss of MtNOD25 does not impair symbiotic nitrogen fixation, indicating that the protein is either non-essential under the conditions tested or functions in a context-dependent manner.
