Flat mites (Tenuipalpidae) are diverse phytophagous arthropods, among which Brevipalpus species are economically important pests capable of transmitting plant viruses. Brevipalpus-transmitted viruses (BTVs) cause localized infections in plants and are classified into two major groups based on cytopathology and genome organization: BTV-C (genera Cilevirus and Higrevirus, family Kitaviridae) and BTV-N (genus Dichorhavirus, family Rhabdoviridae). Despite their significance, the virome of tenuipalpid mite vectors remains poorly characterized. Using high-throughput sequencing (HTS), we analyzed virus populations associated with Brevipalpus and Dolichotetranychus mites collected from multiple plant hosts across two Hawaiian islands. We identified a diverse assemblage of viral sequences affiliated with Kitaviridae, negeviruses, Picornavirales, Narnaviridae, Tombusviridae, Solemoviridae, Ourmiaviridae, Reoviridae, and Potyviridae. Near-complete genomes of citrus leprosis virus C2H and hibiscus green spot virus 2 (both BTV-C) were recovered, highlighting the utility of HTS-based viromics for surveillance of BTVs in mite vectors. In addition, multiple divergent virus-like contigs were identified based on viral hallmark genes and sequence divergence, including Brevipalpus-associated negevirus, Brevipalpus-associated bluner-like virus, and Dolichotetranychus-associated cile-like virus, all showing evolutionary affinities to BTV-C-related viruses. Phylogenetic analyses support evolutionary links between negeviruses and kitavirids, consistent with the hypothesis that Kitaviridae evolved from arthropod-associated ancestors. Although some detected plant viruses may reflect ingestion rather than active replication in mites, this study establishes a robust framework for virome-based surveillance of tenuipalpid mites, advancing our understanding of plant virus evolution and supporting agricultural biosecurity and pest management efforts.Copyright (c) 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Glycerophosphodiester phosphodiesterases (GDPDs) hydrolyze glycerophosphodiesters, phospholipid metabolites generated by acyl hydrolases. We analyzed the function of the 2 related proteins GDPD5 and GDPD6 from Arabidopsis (Arabidopsis thaliana), which are expressed during phosphate starvation and in flower organs. Heterologously expressed GDPD5 and GDPD6 hydrolyzed glycerophosphocholine and other glycerophosphodiesters, indicating that they are involved in the turnover of phosphatidylcholine. The membrane lipid composition in leaves and roots of single gdpd5 or gdpd6 mutants was not altered during growth under normal conditions and phosphate starvation, suggesting that GDPD5 and GDPD6 are not involved in phospholipid turnover under phosphate deficiency. After crossing gdpd5 and gdpd6 null mutants, no double homozygous (g5g5 g6g6) plants were obtained. Genetic analyses of the F1 progeny after selfing, or reciprocal crosses of G5g5 G6g6 with wild-type plants, revealed that the double heterozygous plants cannot produce viable female or male gametes with the haplotypes G5G6 or g5g6. About 50% of the seeds of G5g5 G6g6 plants were aborted. The development of microspores was affected, and ∼50% of pollen grains were distorted and lacked organelles. The anthers of G5g5 G6g6 plants showed decreased phosphatidylcholine content, indicating that GDPD5 and GDPD6 are essential for phosphatidylcholine homeostasis. Therefore, compromised phosphatidylcholine homeostasis affects membrane biogenesis during sporogenesis and gametogenesis, compromising ovule and pollen viability in G5g5 G6g6 plants.

Summary Plants continuously adjust photosynthesis to balance growth and photoprotection under changing environmental conditions. Environmental fluctuations frequently impose a mismatch between energy production and CO2 assimilation. How photochemical reactions are regulated to maintain performance under these conditions remains a central question in plant biology. We previously developed transplastomic tobacco ( Fd1- OE plants) overexpressing ferredoxin (Fd) displaying enhanced photoprotection and growth penalties with a variegated leaf phenotype under greenhouse conditions. Here, we investigate how these plants respond to different growth irradiances using physiological, ultrastructural, and photosynthetic analyses, including PAM, gas exchange, and P700 absorbance measurements, and dynamic-light assays. Fd1 -OE plants progressively recovered growth, leaf phenotype and photosynthetic performance as growth irradiance increased, reaching near WT performance at 1400 μmol m⁻² s⁻¹. This enhanced adaptation to “high-light” was associated with a larger fraction of open PSII reaction centers and enhanced NPQ. Dynamic-light analyses further revealed faster plastoquinone (PQ) turnover, a more oxidized PQ pool and enhanced electron withdrawal downstream of PSI. Our results indicate that Fd overexpression redefines the balance between photochemistry and photoprotection. This adjustment shifts adaptation toward higher irradiance and enhances photosynthetic performance under changing light environments. Electron partitioning downstream of PSI emerges as a promising target to improve photosynthetic resilience. One sentence summary Overexpression of Fd1 in tobacco plants adjusts photosynthesis/photoprotection trade off to enhance high-light adaptation

Awns of wild barley (Hordeum vulgare ssp. spontaneum L.) are rough by default due to silicified upward-oriented trichomes on the awn's epidermis, forming a ratcheted surface, which is advantageous for seed dispersal and burial. Cultivated barley, however, may carry smooth awns covered by smaller barbs or lacking barbs completely. The gene Raw1 on chromosome 5H is a major factor controlling barley awn roughness and was shown to encode a LONG AND BARBED AWN1 (LABA1) homolog. Here we report, by using quantitative analysis of the barb trait, map-based cloning and Cas9-mediated gene knock-out, a second gene Raw7, located on barley chromosome 7H, encoding a putative two-component response regulator. We propose that Raw7 acts downstream of Raw1 in a cytokinin signaling pathway underlying cell cycle control in epidermal barb primordia cells. Raw1 and Raw7 show epistatic interaction, suggesting that Raw1 acts as the primary driver of barb initiation, while Raw7 modulates barb size and frequency. Our findings provide the foundation to study the selection and domestication history of the awn roughness trait in barley, and thus to dissect if awn roughness is providing an advantage in cultivated barley or if the trait persisted after domestication due to linkage drag.

Summary Strigolactones (SLs) are known to regulate shoot architecture and to be involved in plant responses to environmental stress, whereas their specific contributions to drought adaptation in barley remain incompletely defined. In this study, we analysed transcriptional, hormonal, and physiological responses to water deficit in barley SL mutants affected in early biosynthesis ( Hvd10 and Hvd17 ), late biosynthesis ( Hvmax1a ), or signalling ( Hvd14 ). The Hvd10, Hvd17 , and Hvd14 mutants exhibited the typical high-tillering phenotype of SL deficiency, whereas Hvmax1a displayed characteristics similar to the wild type (WT), indicating functional differences within the SL biosynthetic pathway. Transcriptome analysis showed a clear overlap in gene expression among the high-tillering SL mutants under both control and drought conditions. We also used computational methods to identify potential transcription factors that might regulate SL-dependent gene expression. A drought experiment showed that SL mutants exhibited reduced biomass, relative water content, and photosynthetic efficiency, with the most pronounced effects observed in the high-tillering lines. Drought also activated the abscisic acid (ABA) pathway in all genotypes, with particularly high accumulation of ABA metabolites in the high-tillering SL mutants. Notably, Hvmax1a resembled the mutant-like metabolic profile, despite maintaining a wild-type-like architecture. Taken together, these results provide new insights into the roles of SL pathway components in drought responses and highlight functional differences among individual genes influencing both plant architecture and stress-related transcriptional programmes. Furthermore, the mutants generated in this study using Cas9-mediated genome editing represent a valuable genetic collection for future research into SL-mediated development and stress responses in barley.

Spatially confined gene expression determines cell identity and is fundamental to complex plant traits. In the evolutionary transition from C-3 to the more efficient C-4 photosynthesis, restricting the glycine decarboxylase reaction to bundle sheath cells initiates a carbon concentrating mechanism via the photorespiratory glycine shuttle. This evolutionary step is generally thought to play an essential role in the progression from ancestral C-3 to C-4 photosynthesis. Plants operating this shuttle are often referred to as C-3-C-4 intermediates or C-2 species. Within the Brassicaceae family, which includes model and crop plants, such species have evolved independently at least five times. However, research on the biochemistry of C-3-C-4 intermediates in the Brassicaceae has been limited to a few case studies of differentially localized proteins between mesophyll and bundle sheath cells. Here, we leverage recent advances in single-cell transcriptome sequencing to better understand how cellular specialization affects interconnected pathways. We generated a single-nuclei RNA sequencing dataset for Moricandia arvensis, a Brassicaceae with C-3-C-4 intermediate characteristics, and compared it to a publicly available single-cell transcriptome of leaf tissue of the C-3 Arabidopsis thaliana. We confirmed the localization of selected photorespiratory proteins by electron microscopy of immunogold-labelled leaf sections. Our analysis revealed a M. arvensis-specific shift in expression of genes directly associated with the photorespiratory reactions, including components of the glycine decarboxylase complex, glutamate:glyoxylate aminotransferase, and glycolate oxidase, suggesting a shuttle of several C-2 metabolites to the bundles sheath. Additionally, associated pathways, such as ammonium assimilation, synthesis of specific amino acids, redox regulation, and transport, also showed enhanced abundance in the M. arvensis bundle sheath.

Strigolactones (SLs) are a class of plant hormones that play a crucial role in shaping plant architecture, significantly influencing plant adaptation to harsh environmental conditions. In this study, we examined the effects of a mutation in a component of the barley SL signalling pathway, the SL repressor HvDWARF53A, on plant growth and drought tolerance. We compared the results with those of a previously described barley mutant, which is highly tillered and drought-sensitive, carrying a mutation in the SL receptor gene HvDWARF14. The two mutants, hvd14.d and hvd53a.f, displayed contrasting phenotypes, including differences in plant height, tillering, and drought sensitivity. Under control conditions, ultrastructural analysis of hvd53a.f revealed smaller chloroplasts and fewer grana stacks, which may account for its reduced photosynthetic efficiency. Conversely, transcriptomic analysis linked the differentially expressed genes in hvd53a.f to antioxidation and stress responses, suggesting a potentially enhanced capacity to cope with drought. Further analysis revealed a strong connection between the SL signalling pathway and circadian clock components. Among these, CIRCADIAN CLOCK ASSOCIATED 1 emerged as a potential SL-responsive transcription factor (TF), possibly playing a key role in regulating tillering. Under drought conditions, hvd53a.f exhibited enhanced tolerance, as evidenced by higher relative water content, reduced chlorophyll degradation, and stable, albeit reduced, photosynthetic performance. Here, we identified the SL-related TF JUNGBRUNNEN 1 as a potential regulator of genes involved in water deficit response and antioxidation processes. Overall, the hvd53a.f mutation enhances drought tolerance while maintaining low, stable photosynthesis, highlighting HvD53A as a central node connecting SL signalling to stress resilience.

Chloroplasts are the sites of photosynthesis but also host essential metabolic and biosynthetic pathways; therefore, the regulation of the chloroplast population is of crucial importance for the viability of the plant. Chloroplast division is closely linked to leaf development, but the coordination of cell expansion, division, and chloroplast multiplication at the molecular level is still poorly understood. Auxin signalling influences leaf growth and may also mediate chloroplast biogenesis and proliferation. Most studies focused on auxin and the development of chloroplasts in the fruit, emphasising the need for further research on leaf tissue. Overexpression of Growth Regulating Factor 5 (35S:GRF5) increases cell and chloroplast division in Arabidopsis thaliana, resulting in larger leaves with more chloroplasts per cell. In this study, we utilised 35S:GRF5 plants as a model to identify auxin control points that regulate chloroplast division. By examining the impact of changes in auxin homeostasis on chloroplast division and mesophyll cell size and by analysing crosses with selected auxin homeostasis genes, we found that reactive oxygen species-auxin crosstalk influences chloroplast multiplication during the cell expansion phase. Evidence indicates that GRF5 modulates auxin responsiveness by regulating the expression of UDP-glucosyltransferase UGT74E2 and PIN-LIKES3/5, which are key players in intracellular auxin homeostasis. These findings provide potential targets for modulating chloroplast abundance to improve photosynthetic efficiency in crops and highlight key areas for further research.

Variegation mutants provide valuable insights into chloroplast biogenesis. We characterized a newly identified variegated barley mutant, in which the phenotype is controlled by duplicate dominant epistasis-representing, to our knowledge, the first reported case of digenic control in chloroplast-deficient mutants. The causal loci, Var4 and Var5, were mapped on chromosomes 2H and 3H. We used whole-genome resequencing to identify candidate genes. Our two top candidate genes are an NBR1-like selective autophagy receptor gene and a DNAJ-domain containing gene, respectively. Based on their homology-based functional annotation, both candidates could be implicated in chloroplast proteostasis, regulating protein import, folding, and/or degradation. We propose that mild, independent defects in proteostasis from each mutation act synergistically to surpass a functional threshold, impairing chloroplast development in early leaves while allowing partial recovery in later stages. These findings highlight a novel digenic mechanism underlying variegation and point to proteostasis as a central vulnerability in chloroplast biogenesis. ### Competing Interest Statement The authors have declared no competing interest.

A biological invasion of the Coconut Rhinoceros Beetle (CRB; Oryctes rhinoceros) to the island of Oahu was discovered in late 2013, posing a threat to palm trees on the island and potential for accidental export to other Hawaiian Islands and sub-tropical palm growing regions of California and Florida. Delineation of populations by physical trapping in remote, undeveloped areas is a critical part of the program for containment and eradication. Continuous surveillance near ports of entry is especially important to eliminate incipient populations rapidly and mitigate the risk of human-assisted transport. Traditional trap monitoring for the CRB is labor-intensive, costly, and temporally inadequate. We have developed an autonomous trap surveillance system framework using electronic sensors and front and backend remote cloud systems for monitoring the CRB trap contents. The customized surveillance system incorporates a camera and digital microphone, and communicates data through a cellular network using Category-M (CAT-M) Low-Power Wide-Area Network (LPWAN) with an integrated GNSS chip for precise geolocation of catches. Hourly monitoring data from early deployments of the system have demonstrated that adult CRB have a crepuscular behavior, with over two-thirds of catches occurring after sunset within three hours of twilight, and fewer than 1% occurring unambiguously during daylight. The system represents a significant advance for trap monitoring, and can prove valuable for identifying biological behaviors that might be exploited for more effective control.

The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben is a leading international plant science institute specializing in biodiversity and crop plant performance research. Over the last decade, all phases of the research data lifecycle were implemented as a continuous process in conjunction with information technology, standardization, and sustainable research data management (RDM) processes. Under the leadership of a team of data stewards, a research data infrastructure, process landscape, capacity building, and governance structures were successfully established. As a result, a generic research data infrastructure was created to serve the principles of good scientific practice, archiving research data in an accessible and sustainable manner, even before the FAIR criteria were formulated. In this paper, we discuss success stories as well as pitfalls and summarize the experiences from 15 years of operating a central RDM infrastructure. We present measures for agile requirements engineering, technical and organizational implementation, governance, training, and roll-out. We show the benefits of a participatory approach across all departments, personnel roles, and researcher profiles through pilot working groups and data management champions. As a result, an ambidextrous approach to data management was implemented, referring to the ability to efficiently combine operational needs, support daily tasks in compliance with the FAIR criteria, while remaining open to adopting technical innovations in an agile manner.

We used draft mitochondrial genomes (mitogenomes) to explore introduction histories of the destructive coconut rhinoceros beetle (CRB, Oryctes rhinoceros L.) throughout the Pacific and its native range, focusing on re-evaluating the relationship between members of the CRB-G haplotype grouping ( sensu Marshall et al. 2017) as previously assessed by the partial mtCOI (mitochondrial DNA cytochrome oxidase subunit I ) gene. Mitogenome analyses that included historical CRB collections confirmed the 2007 invasive CRB population in Guam was found only in Guam, while there was a detection of a second novel CRB mitogenome, suggesting a new recent introduction(s) into Guam. Further, mitogenome analyses linked: Palau CRB with Indonesia and Philippines native range populations; Papua New Guinea and Solomon Islands CRB with native range Malaysia CRB; Marshall Islands CRB with Solomon Islands CRB; and Samoa and Fiji CRB with Sri Lanka. We therefore provided evidence of historical and current CRB hitchhiking pathways between various native and introduced locations. The results build upon the previous partial mtCOI marker framework to improve the resolution of diversity present within CRB. This study also highlights a need to implement new CRB population nomenclatures based on full mitochondrial DNA genomes.

Centromeres are essential for kinetochore assembly and spindle attachment. While chromosomes of most species are monocentric with a single centromere, a minority exhibit holocentricity, with a centromere along the chromatid length. Sporadic emergence of holocentricity suggests multiple independent transitions. To explore this, we compare the centromere and (epi)genome organization of two sister genera with contrasting centromere types: Chamaelirium luteum with large macro-monocentromeres and Chionographis japonica with holocentromeres. Both exhibit chromosome-wide histone phosphorylation patterns distinct from typical monocentric species. Kinetochore analysis reveals similar chimeric Borealin in both species, with additional KNL2 loss and NSL1 chimerism in Cha. luteum. The broad-scale synteny between both genomes supports de novo holocentromere formation in Chi. japonica. Despite sharing features with both centromere types, macro-monocentromeres do not represent a direct link between mono- and holocentromeres. We propose a model for the divergent evolution involving kinetochore gene mutations, altered histone phosphorylation patterns, and centromeric satellite DNA amplification.

Background Seed retention is the basic prerequisite for seed harvest. However, only little breeding progress has been achieved for this trait in the major forage grasses. The aim of this study was to evaluate the potential of plant genetic resources of the important fodder grasses Festuca pratensis Huds. and Lolium perenne L. as source for seed retention in the breeding process. Furthermore, the morphology of the abscission zone, where shattering occurs, was studied on the cell tissue level in different developmental stages of contrasting accessions. Results 150 and 286 accessions of Festuca pratensis and Lolium perenne were screened for seed retention, respectively. Contrasting accessions were selected to be tested in a second year. We found a great variation in seed retention in Festuca pratensis and Lolium perenne , ranging from 13 to 71% (average: 35%) and 12 to 94% (average: 49%), respectively, in the first year. Seed retention was generally lower in the second year. Cultivars were within the accessions with highest seed retention in Festuca pratensis , but had lower seed retention than ecotypes in Lolium perenne . Field-shattered seeds had a lower thousand grain weight than retained seeds. Cell layers of the abscission zone appeared already in early seed stages and were nested within each other in accessions with high seed retention, while there were two to three superimposed layers in accessions with low seed retention. Conclusions Plant genetic resources of Lolium perenne might be a valuable source for breeding varieties with high seed retention. However, simultaneous selection for high seed weight is necessary for developing successful commercial cultivars.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b 561 domain, as critical to maintain cell elongation and meristem integrity under low P. We demonstrate that HYP1 mediates ascorbate-dependent trans-plasma membrane electron transport and can reduce ferric and cupric substrates in Xenopus laevis oocytes and in planta . HYP1 expression is up-regulated in response to P deficiency in the proximal zone of the root apical meristem. Disruption of HYP1 leads to increased Fe and callose accumulation in the root meristem and causes significant transcriptional changes in roots. We further demonstrate that HYP1 activity overcomes malate-induced Fe accumulation, thereby preventing Fe-dependent root growth arrest in response to low P. Collectively, our results uncover an ascorbate-dependent metalloreductase that is critical to protect root meristems of P-deficient plants from increased Fe availability and provide insights into the physiological function of the yet poorly characterized but ubiquitous CYBDOM proteins.

Camelina is an oil seed crop that is enjoying increasing interest because it has a particularly valuable fatty acid profile, is modest regarding its water and nutrient requirements, and is comparatively resilient to abiotic and biotic stress factors. The regeneration of plants from cells accessible to genetic manipulation is an essential prerequisite for the generation of genetically engineered plants, be it by transgenesis or genome editing. Here, immature embryos were used on the assumption that their incomplete differentiation was associated with totipotency. In culture, regenerative structures appeared adventitiously at the embryos’ hypocotyls. For this, the application of auxin- or cytokinin-type growth regulators was essential. The formation of regenerative structures was most efficient when indole-3-acetic acid was added to the induction medium at 1 mg/L, zygotic embryos of the medium walking stick stage were used, and their hypocotyls were stimulated by pricking to a wound response. Histological examinations revealed that the formation of adventitious shoots was initiated by locally activated cell division and proliferation in the epidermis and the outer cortex of the hypocotyl. While the regeneration of plants was established in principle using the experimental line Cam139, the method proved to be similarly applicable to the current cultivar Ligena, and hence it constitutes a vital basis for future genetic engineering approaches.

Pangenomes are collections of annotated genome sequences of multiple individuals of a species. The structural variants uncovered by these datasets are a major asset to genetic analysis in crop plants. Here, we report a pangenome of barley comprising long-read sequence assemblies of 76 wild and domesticated genomes and short-read sequence data of 1,315 genotypes. An expanded catalogue of sequence variation in the crop includes structurally complex loci that have become hot spots of gene copy number variation in evolutionarily recent times. To demonstrate the utility of the pangenome, we focus on four loci involved in disease resistance, plant architecture, nutrient release, and trichome development. Novel allelic variation at a powdery mildew resistance locus and population-specific copy number gains in a regulator of vegetative branching were found. Expansion of a family of starch-cleaving enzymes in elite malting barleys was linked to shifts in enzymatic activity in micro-malting trials. Deletion of an enhancer motif is likely to change the developmental trajectory of the hairy appendages on barley grains. Our findings indicate that rapid evolution at structurally complex loci may have helped crop plants adapt to new selective regimes in agricultural ecosystems.

Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros, classed as ‘CRB-G (clade I)’, has been the focus of much research since 2007, with reports of invasions into new Pacific Island locations (e.g., Guam, Hawaii, Solomons Islands). For numerous invasive species, inference of invasion biology via whole genome is superior to assessments via the partial mtCOI gene. Here, we explore CRB draft mitochondrial genomes (mitogenomes) from historical and recent collections, with assessment focused on individuals associated within the CRB-G (clade I) classification. We found that all Guam CRB individuals possessed the same mitogenome across all 13 protein-coding genes and differed from individuals collected elsewhere, including ‘non-Guam’ individuals designated as CRB-G (clade I) by partial mtCOI assessment. Two alternative ATP6 and COIII partial gene primer sets were developed to enable distinction between CRB individuals from Guam that classed within the CRB-G (clade I) haplotype grouping and CRB-G (Clade I) individuals collected elsewhere. Phylogenetic analyses based on concatenated ATP6–COIII genes showed that only Guam CRB-G (clade I) individuals clustered together, and therefore Guam was not the source of the CRB that invaded the other locations in the Pacific assessed in this study. The use of the mtCOI and/or mtCOIII genes for initial molecular diagnosis of CRB remained crucial, and assessment of more native CRB populations will further advance our ability to identify the provenance of CRB invasions being reported within the Pacific and elsewhere.

AbstractSpatially confined gene expression determines cell identity and is fundamental to complex plant traits. In the evolutionary transition from C3to the more efficient C4photosynthesis, restricting the glycine decarboxylase reaction to bundle sheath cells initiates a carbon concentrating mechanism via the photorespiratory glycine shuttle. This evolutionary step is generally thought to play an essential role in the progression from ancestral C3to C4photosynthesis. Plants operating this shuttle are often referred to as C3-C4intermediates or C2species. Within the Brassicaceae family, which includes model plants and crops, such species have evolved independently at least five times. However, research on the biochemistry of C3-C4intermediates in the Brassicaceae has been limited to a few case studies of differentially localized proteins between mesophyll and bundle sheath cells. Here, we leveraged recent advances in single-cell transcriptome sequencing to better understand how new cellular specialization affects interconnected pathways. We generated a single-nuclei RNA sequencing dataset forMoricandia arvensis, a Brassicaceae with C3-C4intermediate characteristics, and compared it to a publicly available single-cell transcriptome of leaf tissue of the C3Arabidopsis thaliana. We independently confirmed the localization of selected photorespiratory proteins by electron microscopy of immunogold-labelled leaf sections. Our analysis revealed the shift in expression of genes directly associated with the photorespiratory glycine decarboxylase reaction, but also of related pathways, such as ammonium assimilation, synthesis of specific amino acids, redox regulation, and transport to theM. arvensisbundle sheath. In contrast, the expression of these genes was not restricted to this cell type in the C3plant.HighlightSingle-nucleus RNA sequencing ofMoricandia arvensisreveals bundle sheath cell-specific expression of photorespiration genes and associated pathways beyond glycine decarboxylase, including ammonium assimilation and redox regulation. This highlights the key role of metabolic compartmentalization in supporting C3-C4intermediate photosynthesis.