The Plant Journal

Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, our lab described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid and re-analyzed the two Solanaceae somatic hybrids. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them ([~]80%) were asymmetrical, consistent with the break-induced replication (BIR) pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. BIR is the main pathway of interparental recombination in somatic hybrid mitochondria. Likewise, under the fusion compatibility model of mitochondrial horizontal transfer, foreign mitochondria fuse with those in the recipient cell and their genomes likely recombine via BIR, resulting in the integration and/or loss of mitochondrial DNA. Findings of this study are also relevant to mitogenome editing assays. HighlightWe show that the chimeric mitochondrial genomes of somatic hybrids result from one of the three described homologous recombination pathways (BIR), mimicking the fusion compatibility model for plant HGT.

Aberrant meiosis in plants often leads to aneuploidy, genetic instability, and sterility. This can occur due to several factors, including chromosome misalignment, defective synapsis or environmental factors that may result in unusual genetic combinations in the offsprings. Unusual chromosome behavior during male meiosis in Humulus lupulus is linked to irregular chromosome segregation and genome instability. However, the origin of meiotic instability remains unclear. We analyzed the centromeric landscape of H. lupulus to determine its role in aberrant chromosomal segregation during cell division. Using a combination of bioinformatic, molecular and cytogenetic approaches, we identified new centromeric repeats and revealed two types of centromeric organizations. Cytogenetic localization on metaphase chromosomes confirmed the genomic distribution of major repeat arrays and revealed unique features that contribute to aberrant segregation. Two centromeric types are composed of the major repeats SaazCEN and SaazCRM1 which are further accompanied by chromosome-specific centromeric satellites, Saaz40, Saaz293, Saaz85, and HuluTR120. Chromosome 2 displays unbalanced segregation during the cell division, implicating an important role for its centromere structure in segregation patterns. Moreover, Saaz293 is a new marker for studying aneuploidy in hop. Our findings provide new insights on chromosome segregation in hop and highlight the diversity and complexity of the centromere organization in H. lupulus.

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for posttranslational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans. Until now, the regulatory mechanisms underlying Dol accumulation remain elusive. In this report, we have analyzed the natural variation of the accumulation of Dols and six other isoprenoids between 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone, and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis - the AT2G17570 gene encoding a long searched for cis-prenyltransferase (CPT3) and the AT1G52460 gene encoding an alpha-beta hydrolase (ABH) - is experimentally confirmed. These data will help to generate Dol-enriched plants which might serve as a remedy for Dol-deficiency in humans.

Sustainability of maize cultivation would benefit tremendously from early sowing but is hampered by low temperatures during early development in temperate climate. We show that allelic variation of subunit M of NADH-dehydrogenase-like (NDH) complex (ndhm1), discovered in a European maize landrace affects several quantitative traits relevant during early development in cold climates through NDH-mediated cyclic electron transport (CET) around photosystem I, a process crucial for photosynthesis. Starting from a genome-wide association study (GWAS) for maximum potential quantum yield of photosystem II in dark-adapted leaves (Fv/Fm) we capitalized on large phenotypic effects of a hAT transposon insertion in ndhm1 on quantitative traits early plant height (EPH), Fv/Fm, chlorophyll content and cold tolerance caused by reduced protein levels of NDHM and associated NDH components. Analysis of the native allelic series of ndhm1 revealed a rare allele of ndhm1 which is associated with small albeit significant effects on maximum potential quantum yield of photosystem II in dark- and light adapted leaves (Fv/Fm, {Phi}PSII) and early plant height compared to common alleles. Our work showcases the extraction of novel, favorable alleles from locally adapted landraces, offering an efficient strategy for broadening the genetic variation of elite germplasm by breeding or genome editing.

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 MORF2s role in chloroplast RNA maturation. Significance StatementWe 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 eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of five multiprotein complexes (complexes I to V). Analyses of the OXPHOS system by native gel electrophoresis revealed an organization of OXPHOS complexes in supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mFDX-like). This protein was previously found associated with complex I, being part of the bridge domain linking the matrix and membrane arms of the complex. A phylogenetic analysis suggests that the Arabidopsis thaliana mFDX-like evolved from classical mitochondrial ferredoxin, but it lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster essential for the electron transfer function of ferredoxins. Accordingly, our biochemical study shows that AtmFDX-like does not bind an Fe-S cluster, and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9 approach. These lines do not show any growth phenotype under standard growth condition. However, the characterization of the OXPHOS system demonstrates that mFDX-like is important for the assembly of complex I, and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III to form supercomplexes.

Typical C4 plants such as maize possess highly optimized Kranz-type leaf anatomy, whereby concentric wreaths of mesophyll and bundle sheath cells surround closely spaced veins. The veins and the cells that surround them are derived from the middle ground meristem (mGM) through processes that are as yet undefined. Here we distinguished the active zone of vascular development within early leaf primordia, and used comparative transcriptomics of sub-sectioned maize and rice primordia to identify cohorts of genes likely involved in early Kranz development. Leveraging single-nucleus RNA sequencing (snRNA-seq) we then explored the cell heterogeneity and developmental trajectories within single maize leaf primordia. Assisted by in situ hybridization, cell clusters of mGM and procambium were identified, with candidate marker genes showing different yet inter-related expression patterns. Localization of the vascular marker ZmSHR1 was preceded by that of ZmEREB161 and ZmEREB114 in terms of procambium initiation. Potential subclusters of bundle sheath cells and different layer of mesophyll cells were depicted from developing cells toward the tip of sub-sectioned maize primordia. Collectively our results identify potential mGM derived or procambium localized Kranz regulators and provide resources for investigating leaf vein development in maize and rice, at sub-primordium and single-cell resolution.

Gene duplication and structural rearrangements have played a pivotal role in plant genome evolution, enabling functional divergence and the development of novel traits. In soybeans (Glycine max), the I locus, which regulates seed color through tissue-specific subfunctionalization of sRNA loci, offers an example of these mechanisms. Using long-read sequencing, we investigated structural variations underlying four major alleles (I, ii, ik, and i) at the I locus. Our study revealed that large-scale rearrangements, including duplications, inversions, and deletions, within a 180-kb CHS repeat-rich region drive allele-specific RNA silencing through small interfering RNAs (siRNAs). The dominant I allele contains a cis-regulatory region of DnaJ upstream of CHS genes, while the ii and ik alleles feature subtilisin- and P450-driven siRNA loci, respectively. The recessive i allele lacks siRNA production due to deletions or inversions disrupting CHS gene clusters. Phenotypic analyses and RNA-seq confirmed allele-specific, tissue-dependent expression of siRNAs correlating with seed coat pigmentation patterns. This study highlights the evolutionary role of repeat-rich regions in generating regulatory innovations and phenotypic diversity. Our findings underscore the importance of structural rearrangements in domestication traits and demonstrate the power of long-read sequencing for resolving complex genomic regions, advancing both evolutionary biology and crop improvement. SIGNIFICANCE{square} Naturally occurring mutations in the soybean seed coat have revealed the evolution of sRNA genes and their subfunctionalization, resulting in tissue-specific gene expression. {square}Non-homologous recombination of repetitive sequences drives cis-regulatory region rearrangements, leading to the subfunctionalization of sRNA loci that regulates one of domestication traits.

Non-photochemical quenching (NPQ) is an important photoprotective process in plants, but all molecular details of the process(es) involved are yet not understood. We have used advanced spectroscopic techniques (including simultaneous time- and spectrally-resolved room-temperature chlorophyll fluorescence analysis and spectro-kinetic deconvolution) to analyse the processes in Arabidopsis, hybrid aspen and Scots pine plants. We used four well-characterized Arabidopsis lines (npq1, npq2, npq4 and L17) affected in NPQ, together with hybrid aspen lines with corresponding modifications that we generated. The data are described best by a model for NPQ induction with up to five fluorescence components representing distinct biochemical entities. A dominant fluorescing species at the end of NPQ induction was identified as functionally detached and quenched LHCII but most importantly we believe that one represents a "PSII-PSI (Photosystem II-Photosystem I) complex" where direct energy transfer between PSII and PSI (spillover) take place. This provides strong quenching in all three plant species. We suggest a new integrated model for NPQ in higher plants where spillover is a major element and suggest roles for PsbS and zeaxanthin. Moreover, we discuss the link between NPQ and thylakoid rearrangements as thylakoid destacking facilitates direct contact between PSII and PSI; a prerequisite for spillover.

The critically important YUCCA (YUC) gene family is highly conserved and specific to the plant kingdom, primarily responsible for the final and rate-limiting step for indole-3-acetic acid (IAA) biosynthesis. IAA is an essential phytohormone, involved in virtually all aspects of plant growth and development. In addition, IAA is involved in fine-tuning plant responses to biotic and abiotic interactions and stresses. While the YUC gene family has significantly expanded throughout the plant kingdom, a detailed analysis of the evolutionary patterns driving this diversification has not been performed. Here we present a comprehensive phylogenetic analysis of the YUC family, combining YUCs from species representing key evolutionary plant lineages. We identify and hierarchically classify the YUC family into six distinct classes and 41 subclasses. YUC diversity and expansion is explained in the context of protein sequence conservation, as well as spatial and gene expression patterns. The presented YUC gene landscape offers new perspectives on the distribution and evolutionary trends of this crucial family, which facilitates further YUC characterization within plant development and response to environmental change. Short summaryComprehensive phylogenetic and sequence analysis of the YUC gene family presents new insights into factors driving evolutionary diversification. HighlightsO_LIA phylogeny-based classification system for the YUCCA gene family is presented C_LIO_LIYUCCA evolution and structural diversification is described, supporting a fine-tuned spatial and temporal control of auxin biosynthesis, but also holds potential for alternative capabilities C_LI

In contrast to their conserved mammalian counterparts, plant long interspersed nuclear elements (LINEs) are highly variable, splitting into many low-copy families. Curiously, LINE families from the RTE clade retain a stronger sequence conservation and hence reach higher copy numbers. The cause of this RTE-typical property is not yet understood, but would help clarifying why some transposable elements are removed quickly whereas others persist in plant genomes. Here, we bring forward the first detailed study of RTE LINE structure, diversity and evolution in plants. For this, we argue that the Nightshade family is the ideal taxon to follow the evolutionary trajectories of RTE LINEs, given their high abundance, recent activity and partnership to non-autonomous elements. Using bioinformatic, cytogenetic and molecular approaches, we detect 4029 full-length RTE LINEs across the Solanaceae. We finely characterize and manually curate a core group of 458 full-length LINEs in allotetraploid tobacco, show amplification after polyploidization, and trace hybridization events by RTE LINE composition of parental genomes. Finally, we reveal the role of the untranslated regions (UTRs) as causes for the unique RTE LINE amplification and evolution pattern in plants: On one hand, we detect a highly conserved motif at the 3 UTR, suggesting strong selective constraints acting on the RTE terminus. On the other hand, we observed successive rounds of 5 UTR cycling, constantly rejuvenating the promoter sequences. This interplay between exchangeable promoters and conserved LINE bodies and 3 UTR likely allows RTE LINEs to persist and thrive in plant genomes.

Maintaining energy homeostasis is a major challenge for plants in the current context of climate change. The Sucrose-non fermenting 1 (SNF1)-related kinase 1 (SnRK1) complex, a member of the SNF1-AMP-activated protein kinase (AMPK)-SnRK1 family of kinase complexes, is a central player in the regulation of cell energy homeostasis. The -subunit of the complex, which possesses kinase activity and is known as SnRK1.1 or KIN10, plays a role in sensing energy status and coordinating metabolic reprogramming to counter any energy imbalance. The discovery of a dual and dynamic intracellular distribution of SnRK1.1 suggests that the activity and function of SnRK1 might be regulated by spatiotemporal changes. To investigate the spatiotemporal distribution of SnRK1.1, we developed a protocol to quantify its intracellular distribution using fluorescence confocal images acquired along the z-axis in plants expressing SnRK1.1-eGFP. Using the open-source software Fiji/ImageJ, we calculated the ratio between nuclear and non-nuclear SnRK1.1 fractions and defined this as the N/ER index. We validated our method by analyzing the response of SnRK1.1 to photosynthesis inhibition by DCMU, including changes in protein levels and phosphorylation status. In addition, comparison with results obtained using a commercial software-based approach confirmed the compatibility of the N/ER index with different segmentation and quantification tools. Originally designed for leaf tissue images, this protocol can be broadly applied to assess the role of intracellular spatiotemporal changes in a wide range of kinases or fluorescently tagged recombinant proteins. Finally, SnRK1.1 intracellular distribution may also serve as a proxy to assess changes in cellular energy status. One sentence summaryNew method to track SnRK1.1 distribution and changes in plant cell energy status

Histone chaperones mediate assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-functions of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1(CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. Altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage clearly demonstrate the severity of CAF-1 dysfunction. How histone chaperone molecular networks change without a functional CAF-1 remains elusive. Here we present an intriguing observation that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) supresses FAS1 loss-of function. The quadruple mutant fas1nap1;1-3 shows wild-type growth and decreased sensitivity to genotoxic stress. Chromatin of fas1nap1;1-3 plants is less accessible to micrococcal nuclease and progressive loss of telomeres and 45S rDNA is supressed. Interestingly, the strong genetic interaction between FAS1 and NAP1 does not occur via direct protein-protein interaction. We propose that NAP1;1-3 play an essential role in nucleosome assembly in fas1, thus their disruption abolishes fas1 defects. Our data altogether reveal a novel function of NAP1 proteins, unmasked by CAF-1 dysfunction. It emphasizes the importance of a balanced composition of chromatin and shed light on the histone chaperone molecular network.

The enzymatic steps involved in O_SCPLOWLC_SCPLOW-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and O_SCPLOWLC_SCPLOW-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and O_SCPLOWLC_SCPLOW-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and O_SCPLOWLC_SCPLOW-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-O_SCPLOWLC_SCPLOW-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

Plant growth and development face constant threat from various environmental stresses. Transcription factors (TFs) are crucial for maintaining balance between plant growth and defense. Trihelix TFs display multifaceted functions in plant growth, development, and responses to various biotic and abiotic stresses. Here, we explore the role of a trihelix TF, ZmGT-3b, in regulating the growth-defense tradeoff in maize (Zea mays). ZmGT-3b is primed for instant response to Fusarium graminearum challenge by implementing a rapid and significant reduction of its expression to suppress seedling growth and enhance disease resistance. ZmGT-3b knockdown led to diminished growth, but improved disease resistance and drought tolerance in maize seedlings. In ZmGT-3b knockdown seedlings, the chlorophyll content and net photosynthetic rate were strongly reduced, whereas the contents of major cell wall components, such as lignin, were synchronically increased. Correspondingly, ZmGT-3b knockdown specifically downregulated photosynthesis-related genes, especially ZmHY5 (encoding a conserved central regulator of seedling development and light responses), but synchronically upregulated genes associated with secondary metabolite biosynthesis and defense-related functions. ZmGT-3b knockdown induced defense-related transcriptional reprogramming and increased biosynthesis of lignin without immune activation. These data suggest that ZmGT-3b is a regulator of plant growth-defense tradeoff that coordinates metabolism during growth-to-defense transitions by optimizing the temporal and spatial expression of photosynthesis- and defense-related genes. One-sentence summaryZmGT-3b regulates photosynthesis activity and synchronically suppresses defense response.

Whether Photosystem I (PSI) core subunits accumulate prior to light exposure in developing angiosperm seedlings remains unresolved, with conflicting reports across species. Here, we investigated the presence and membrane colocalization of the PSI core subunit PsaA in etioplasts of dark-grown angiosperms representing dicot and monocot species. Immunoblotting showed that PsaA accumulates in etioplasts of all three dicot species examined (pea, Arabidopsis, and runner bean), whereas in the monocot oat it was detected only after prolonged etiolation, at substantially lower levels and with an anomalously high apparent molecular weight. Blue-native PAGE analysis reveals that a fraction of PsaA co-migrates with LPOR, PsaB, FNR, and chlorophyll synthase, suggesting co-localization within a shared membrane microdomain rather than stable complex formation. The thylakoid insertase Alb3 was more abundant in dicot etioplasts, consistent with a potential role in the early integration of PsaA into the membrane. Upon illumination, pea reached PSI functionality faster than oat, with P700 oxidation detectable 30 min earlier, linking the dark accumulation of PsaA to an accelerated photosynthetic onset. These findings demonstrate light-independent accumulation of a PSI core subunit in a species-dependent manner and point to early steps in PSI biogenesis that precede full photosynthetic complex assembly. Highlight Contrary to prevailing models, a Photosystem I core subunit PsaA accumulates in dark-grown angiosperm seedlings before light exposure, revealing light-independent early steps in photosynthetic complex biogenesis.

Acetylation in plant cell walls, including lignin, plays a critical role in biomass processability. High acetate content facilitates uncatalyzed, additive-free hot-water or steam hydrolysis, releasing acetic acid without the need for mineral acids or other chemical pretreatments. Despite the prevalence of acetylation in certain plant species, the genes and enzymes responsible for lignin and xylem acetylation remain largely uncharacterized. Here, we report the identification and functional characterization of an acetyl-CoA:monolignol transferase gene in Populus. Overexpression of this gene in transgenic poplars led to elevated acetate levels in lignin without negatively impacting plant growth or development. Acetate accumulation was positively correlated with gene expression, and among 18 independent transgenic lines, several showed at least a sevenfold increase compared to wild-type controls. This work establishes a genetic basis for lignin acetylation and provides a promising strategy to engineer biomass with improved pretreatability, enhancing the efficiency of biofuel and bioproduct production.

Arabidopsis thaliana Col-0 has plastid and mitochondrial genomes encoding for over one hundred proteins and several ORFs. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of such RNA editing at the protein level is largely unexplored due to the complexities of detection. This study first assembled the non-redundant set of identifiers, their correct protein sequences, and 452 predicted non-synonymous editing sites of which 56 are edited at lower frequency. Accumulation of edited and/or unedited proteoforms was then determined by searching [~]259 million raw MSMS spectra from ProteomeXchange as part of Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). All mitochondrial proteins and all except three plastid-encoded proteins (NDHG/NDH6, PSBM, RPS16), but none of the ORFs, were identified; we suggest that all ORFs and RPS16 are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical challenges of detection.167 predicted edit sites were detected at the proteome level. Minor frequency sites were indeed also edited at low frequency at the protein level. However, except for sites RPL5-22 and CCB382-124, proteins only accumulate in edited form (>98 -100% edited) even if RNA editing levels are well below 100%. This study establishes that RNA editing for major editing sites is required for stable protein accumulation.

In eukaryotic organisms, proper chromosome segregation during cell division depends on the centromeric histone H3 (CENH3) variant. Our previous studies identified a plant CENH3 assembly factor, Kinetochore Null2 (KNL2), that possesses a centromere-targeting motif, CENPC-k, similar to the CENPC motif in CENP-C. Additionally, we have demonstrated that KNL2 can bind DNA in vitro, independent of its CENPC-k motif. Thus, the mechanism underlying the binding of KNL2 to centromeric DNA remains elusive. Our study shows that the CENPC-k and CENPC motifs alone are not sufficient to target the centromere in N. benthamiana and A. thaliana. In-silico analysis revealed flanking DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their importance in interacting with centromeric DNA. Fusion of protein fragments containing these motifs to EYFP facilitated targeting to the centromere. Deletion of DNA-binding domains reduced the centromeric localization of KNL2-C, whereas fusion of CENPC-k to the H-NS protein from E. coli targeted it to centromeres. We conclude that targeting of KNL2 and CENP-C proteins to centromeres is dependent on the CENPC-k/CENPC motifs, and their sequence-independent DNA-binding promotes anchoring at the centromere. Understanding the targeting mechanisms of KNL2 and CENP-C may help to engineer kinetochore structure by targeting chromatin modifying proteins to centromeres.

Communication across different plant cell compartments relies on an intricate network of molecular interactions, required for the orchestration of organelle development and adaptation to the environment. In this scenario, the Pentatricopeptide Repeat (PPR) Protein GENOMES UNCOUPLED1 (GUN1) plays a key role in transferring information from both developing and mature chloroplasts to the nucleus with the aim to coordinate gene expression between the two genomes. However, its role and the related signaling molecules are still under debate. To help shed light on this matter, we attempted the holistic description of Arabidopsis thaliana proteome upon perturbation of chloroplast biogenesis by lincomycin (Lin), in a genetic context devoid of GUN1-dependent plastid-to-nucleus signaling pathway. Furthermore, the topological analysis of protein-protein interaction (PPI) and protein co-expression networks allowed the identification of protein hubs/bottlenecks characterizing genotypes and conditions, such as proteases, HSPs/Chaperones and redox proteins. Taken together, our findings indicate that GUN1 is required to orchestrate a plastid-located response to plastid protein synthesis inhibition while, in its absence, the reorganization of the activities associated with extra-plastid compartments, such as cytosol, vacuole and mitochondria, prevails. From this landscape, we documented a new role of the Oxygen Evolving Complex subunit PsbO, which appears to be an unconventional photosynthetic protein, as it accumulates in non-photosynthetic plastids and plays a central role in promoting chloroplast breakdown when plastid functions are altered.