The Plant Cell

The chloroplast is a semi-autonomous organelle with a compact genome encoding ~100 expressed genes. Numerous and widespread promoters coupled with inefficient transcription termination leads to a highly complex primary transcriptome that undergoes extensive RNA quality control orchestrated by RNA binding proteins and ribonucleases. Among the ribonucleases, only RNase J (RNJ) is absolutely required for plant germination and survival, likely because its deficiency leads to massive accumulation of antisense RNA which inhibits translation. As one approach to understanding the selectivity of RNJ for antisense RNA and noncoding regions, we created Arabidopsis lines overexpressing catalytically inactive versions of the enzyme designed to trap its substrates. Initially, transgenic plants appeared phenotypically normal. However, crosses used to generate homozygous transgenic lines yielded progeny that developed varying degrees of chlorosis within 2-3 weeks. Concomitantly, these plants exhibited chloroplast transcriptome alterations similar to RNJ-silenced lines, along with mRNA changes consistent with retrograde signaling. This chlorosis could be correlated to the likely decay of RNJ protein, but not its endogenous transcript. The result could be replicated by attempting to overexpress a wild-type version of RNJ, in which case even one transgene copy was sufficient to induce chlorosis. Our results suggest that RNJ is under protein expression level surveillance to prevent wholesale destruction of the plastid transcriptome.

Understanding how photosynthetic organisms acclimate to excess light is essential for the development of robust chassis for synthetic biology approaches aimed at expanding the photosynthetically active absorption spectrum. High-light (HL) tolerant strains were previously generated by laboratory evolution from a laboratory type (LT) strain of Synechocystis sp. PCC 6803, with tolerance attributed to a small number of specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroduction of these mutations into the LT background was sufficient to confer HL tolerance. However, the mechanisms by which a limited set of point mutations mediates HL tolerance have remained unclear. Here, integrated transcriptomic and proteomic analyses of HL-tolerant strains reveal a coordinated network of responses underlying HL tolerance. The NdhF1F124L mutation increased the accumulation of NDH-1 complex subunits, likely accounting for the previously observed enhancement of cyclic electron flow. In contrast, EF-G2R461C increased the abundance of multiple functional classes of proteins associated with HL tolerance, while post-transcriptionally reducing the level of the phycobilisome linker protein CpcC2, resulting in a decreased antenna size. Integrated analyses further demonstrated that HL tolerance involves transcriptional regulation of protein abundance, including the maintenance of phosphate metabolism. Consistently, overexpression of two genes from the Pho regulon increased HL tolerance. Overall, this study demonstrates how a small number of point mutations in genes with central cellular functions can reprogram the cyanobacterial cell to achieve enhanced tolerance to HL.

SUMOylation is an essential and evolutionarily conserved post-translational modification that enables rapid cellular adaptation to environmental stress. Despite its importance, how the SUMOylation machinery controls its dynamics remains poorly understood. Here, we integrated a mathematical model of the plant SUMO cycle with quantitative imaging and biochemical data to decode the regulatory logic of SUMOylation dynamics. The model reveals a fundamental principle: steady-state SUMOylation levels are determined not by the full complement of SUMO-cycle components, but by the abundance of SUMO modifier proteins and E1 enzymes, together with SUMO protease abundance and activity. Strikingly, when we integrated fluorescence measurements from reporters for SUMO-cycle components in Arabidopsis roots after three hours of abiotic stress, the model predicted decreased SUMOylation levels across several tissues, contrary to prevailing evidence. Motivated by this unexpected prediction, we performed time-resolved imaging to better characterize the dynamics of key SUMO-cycle components after salt and osmotic stress. These data revealed highly dynamic component levels within the first three hours of stress that were not captured in the previous measurements. Incorporating this temporal data into the model uncovered a twin-peaked SUMOylation response to salt stress: an early surge underpinned by protease dynamics, followed by a decline and a second peak driven by elevated SUMO modifier and E2 levels. Western blot analysis revealed a corresponding twin-peaked pattern of SUMOylation. Together, our findings demonstrate that the plant SUMO cycle is a highly dynamic system that achieves tissue- and stress-specific control of SUMOylation through the coordinated interplay of multiple SUMO-cycle components.

Poplar (Populus spp.) is a model for tree biology and a platform for engineering woody biomass, biofuels, biomaterials, and bioproducts. Many relevant traits depend on tissue position, developmental stage, and cell type, yet these spatial relationships are difficult to recover from bulk or single-cell transcriptomes. Here, we generated a spatial transcriptome atlas of Populus tremula x P. alba INRA 717-1B4 across the shoot apex, axillary bud, stem, and petiole. After quality control, the atlas retained 29,687 spatial spots from 45 tissue sections and detected 58,748 genes. Histology-guided clustering and marker analysis resolved meristematic, epidermal, cortical, vascular, and organ-specific domains. Cross-organ comparisons assessed whether published markers retained tissue-associated expression across different anatomical contexts and developmental stages, while de novo analysis identified additional domain-enriched candidates. As case studies of the utility of the atlas, we examined the emergence of trichome-associated programs in the shoot apex and adaxial - abaxial expression differences in petioles. A trichome identity score based on poplar markers from the single-cell shoot atlas peaked along the inferred meristem-to-primordium trajectory, revealing spatially localized expression of trichome-associated programs during early leaf development. Petiole expression differences were concentrated in the epidermis and cortex and involved polarity-associated, auxin-responsive, and cell-wall-remodeling genes, with distinct expression profiles across leaf positions. Together, these data provide a spatial reference for investigating tissue differentiation and developmental patterning in a transformable poplar genotype.

Rhamnogalacturonan I (RG-I) is a major pectin domain and an important wall component for cell-cell adhesion. Arabidopsis seed mucilage serves as a powerful model to identify and characterize pectin-related enzymes, since this gelatinous capsule is composed predominantly of RG-I. Although multiple glycosyltransferase (GT) families participate in pectin biosynthesis, only a limited number of their members have been functionally characterized in vivo or biochemically characterized in vitro. In this study, we characterized the biological functions of PEELING WALLS1 (PEEL1), a Golgi-localized GT106 protein that is related to known RG-I rhamnosyltransferases (RRTs). Knocking out PEEL1, but not its close paralog PEEL1-LIKE (PEEL1L), led to patchy mucilage release from seeds upon hydration. However, RG-I content was not compromised in mucilage extracted from peel1 or peel1 peel1L mutant seeds. Treatment of seeds with a cation chelator restored mucilage expansion but failed to rescue the underlying defect in epidermal cell adhesion. Histological experiments and transgene complementation demonstrated that optimal expression of PEEL1 is required for the adhesion of primary walls to the seed surface. Collectively, these findings identify PEEL1 as a previously unrecognized contributor to primary cell wall adhesion and suggest that different RRT-related proteins contribute to unique pectic structures or localizations in vivo, rather than total mucilage RG-I abundance.

Cellulose synthase complexes build the load-bearing cellulose microfibrils of plant cell walls, yet how the abundance of their catalytic CELLULOSE SYNTHASE A (CESA) subunits is maintained remains unclear. Here, we used multi-bait TurboID proximity labelling with ten cellulose-synthesis-associated baits and six subcellular controls to define a high-confidence cellulose synthase neighbourhood. Stringent spatial and recurrence-based filtering yielded a core network of 119 interactions among 44 proteins and identified three members of the conserved Bcl-2-associated athanogene (BAG) family as previously unrecognised regulators of cellulose synthase homeostasis. BAG1-3 associated with primary-wall CESAs in reciprocal proximity-labelling experiments. Arabidopsis bag mutants showed reduced cellulose accumulation, hypersensitivity to cellulose-synthesis inhibitors, and markedly decreased CESA protein abundance without corresponding changes in CESA transcript levels. Loss of BAG function also increased the accumulation of CESA6 in vacuolar compartments. These findings identify BAG proteins as previously unrecognised regulators of cellulose synthase homeostasis and link a conserved proteostasis-associated protein family to plant cell wall biosynthesis. More broadly, the study establishes multi-bait proximity labelling, combined with cell location-specific controls, as a strategy for resolving dynamic protein networks whose components traffic through multiple subcellular compartments.

Plastids form stroma-filled tubular extensions, termed stromules, that are implicated in inter-organellar communication. In pathogen-challenged Nicotiana benthamiana, stromule formation is driven by kinesins acting along microtubules. However, whether this mechanism also operates in unstressed plants or in other species remains unknown. Using systematic genetic analysis of all class XI myosin genes and quantitative time-lapse imaging in Arabidopsis (Arabidopsis thaliana), we show that basal stromule formation is predominantly myosin dependent through two distinct mechanisms. Myosin XI-I generates most stromules by driving oscillatory nuclear movement that mechanically pulls stromules from stationary plastids within the approx. 8m wide stromule-promoting zone. Myosins XI-K, XI-1, and XI-2 additionally drive a movement-independent, nucleus-associated stromule subpopulation within the same zone, establishing two mechanistically distinct pathways. We propose that movement-coupled stromules function as dynamic tethers maintaining plastid-nucleus proximity during nuclear oscillations, while movement-independent stromules expand the plastid membrane area within the perinuclear space, increasing the potential contact surface with the nucleus and surrounding environment. Together, both populations are proposed to enhance signal and metabolite exchange at the plastid-nucleus interface. These findings indicate that stromule biogenesis is both mechanistically and functionally context dependent, challenging the universality of the kinesin-microtubule model across plant species and physiological states.

The functional roles of glycans attached to the proline (Pro) rich region of arabinogalactan proteins are largely elusive. We have tested the possibility of type II arabinogalactans acting as protein fate determinants for the FASCICLIN LIKE ARABINOGALACTAN PROTEIN 4 (FLA4). Pro-to-Ala substitutions in the carboxy-terminal region caused in planta protein destabilization in an ECHIDNA-dependent trans-Golgi compartment and plasma membrane shedding in an aspartate protease-dependent manner. The introduction of diverse glycomodules to the FLA4 carboxy-terminus counteracted the stability loss, with AGP type glycosylation motifs more efficient than N-glycosylation signals and extensin structures. The resistance of FLA4 stability and localization to genetic interference with O-glycosylation suggested that additional protein properties, such as intrinsic disorder and secondary structure were crucial for FLA4 stability. Consistently, disorder-maintaining site directed mutants maintained FLA4 stability in a glycosylation-independent manner. Our data support a role for proline-rich regions in determining protein fate by generating intrinsically disordered domains in the peptide chain and providing acceptor sites for O-glycosylation.

Root nodule symbiosis allows legumes to meet their nitrogen requirements. However, the causes of symbiosis failure at high temperatures remain unknown. We demonstrated that elevated temperatures affect both the host and the microbial partner in Medicago truncatula and chickpea. Rhizobial viability and nod gene expression persisted under heat stress, but plant transcriptome analysis revealed a strong downregulation of the nodule organogenesis machinery. Nodule organogenesis was restored by constitutive overexpression of NIN-LBD16-NFYA1-NFYB16, miR2111, and CLE5 knockdown but failed to restore nitrogen fixation. In contrast, the genetic manipulation of the competence pathway involving CEP peptides significantly improved both nodulation and nitrogen fixation. Constitutive loss of the autoregulation of nodulation (AON) pathway in the SUNN alleles also rescued root nodulation and nitrogen fixation. Therefore, heat stress perturbs the balance between AON and the competence pathway, and genetic manipulation of these pathways is a promising approach to engineer thermotolerant nitrogen fixation in crop legumes.

Jasmonates are essential phytohormones that coordinate defense responses and developmental programs across land plants. In angiosperms, the active jasmonate ligand jasmonoyl-L-isoleucine (JA-Ile), is produced through GH3-mediated conjugation of jasmonic acid to isoleucine and JA-Ile homeostasis is further shaped by ILR1/ILL-family amidohydrolases. In contrast, the primary bioactive jasmonate ligand in bryophytes, dinor-12-oxo-phytodienoic acid (dn-iso-OPDA), is inactivated through conjugation with amino acids, raising the question of whether these conjugates constitute a reversible hormone reservoir or an irreversible catabolic end point. Although the ILR1-like family has been characterized extensively for its role in auxin and jasmonate homeostasis in angiosperms, its function in bryophytes remains basically unexplored. Here we show that MpILR1, the sole Marchantia ortholog of the ILR1/ILL family, hydrolyzes a specific subset of dn-iso-OPDA-amino acid conjugates in vivo. Loss-of-function Mpilr1 mutants exhibit enhanced accumulation of dn-iso-OPDA conjugated to hydrophobic amino acids (Val, Leu and Ile) but not to hydrophilic residues (His, Glu and Gln), demonstrating substrate-selective hydrolysis. MpILR1 hydrolytic activity is required for full dn-iso-OPDA-mediated responses, including transcriptional activation and defense against gastropod herbivory. These findings establish MpILR1 as a key positive regulator of jasmonate signaling in Marchantia polymorpha and suggest that hormone conjugation/deconjugation is an ancient regulatory mechanism evolved during plant terrestrialization.

Zea mays (maize) flowering time is genetically determined and a critical yield determinant. Yet mechanistic understanding of maize flowering remains poor. Indeterminate1 (Id1), a zinc-finger transcription factor (TF), is a monocot-conserved master regulator of maize flowering. Epistasis between Id1 and the ZeaCentroradialis-Delayed Flowering1 (Zcn-Dlf1) inductive pathway partly explains ID1 floral control; however, the strong mutant id1- floral delay is not explained by this pathway alone. To better characterize Id1 actions, we performed single-cell assay for transposase-accessible chromatin and single nucleus RNA sequencing (scATAC-seq and snRNA-seq) comparing Id1+ and id1- developing leaves. These analyses reveal id1- chromatin remodeling via TEOSINTE BRANCHED1 CYCLOIDEA PROLIFERATING CELL FACTOR (TCP) and APETALA2/ETHYLENE RESPONSEFACTOR (AP2/ERF) transcription factors and provide candidate direct targets that include AP2/ERF genes. These candidate direct targets include the family of {beta}-glucosidase genes that lose expression in id1-. Unexpectedly, CRISPR/Cas9 {beta}-glucosidase edits produced plants that phenocopied terminal ear1- (te1-) mutants. This phenocopy prompted an investigation into the genetic relationship between id1-, te1- and flowering. Surprisingly, id1- te1- plants exhibited a synergistic floral delay, producing ~90 leaves before inflorescence production. Beyond highlighting hitherto unappreciated Te1 autonomous flowering roles, this genetic synergy raises the hypothesis that meristem leaf primordia cessation underpins maize flowering.

Local metabolic programs can determine whether developing organs maintain growth, differentiate, or degenerate. However, the spatial establishment of such programs during reproductive development remains poorly understood. In cereal inflorescences, this question is closely tied to grain number because the fate of initiated floral primordia is shaped by the developmental position, metabolic support, genotype, and environmental stress. To understand the metabolic logic underlying floral fate in barley (Hordeum vulgare L.), we mapped the spatial distribution of metabolites across the inflorescence in wild-type Bowman and in hvcmf4, a mutant that undergoes premature apical inflorescence degeneration. Amino acid-, carbohydrate-, and chlorophyll-associated metabolites form clear developmental gradients along the inflorescence axis. These gradients are progressively remodeled during developmental pre-anthesis tip degeneration but are both accelerated and spatially distorted in hvcmf4. Within this broader metabolic landscape, two discrete domains stand out. First, spermidine-associated domains specifically marked reproductive meristems, a pattern corroborated by the meristem-enriched expression of polyamine biosynthetic genes. This polyamine-rich meristem state declined as developmental tip degeneration proceeded and was prematurely lost in hvcmf4, where polyamine metabolism appeared to shift toward oxidative breakdown via the polyamine oxidase pathway. Second, lysophosphatidylcholine accumulated specifically at floral attachment and vascular supply zones. The developmental reduction of this bioactive lysophospholipid-associated domain in the apical regions, and its stronger disruption in hvcmf4, coincided with the reprogramming of lipid-remodeling and auxin transport-associated genes, suggesting impaired coordination of lipid signaling, auxin canalization, and vascular support. Together, our study reveals spatial polyamine and lysophospholipid domains that couple meristem maintenance with vascular support, providing a metabolic framework for the fate of developing cereal inflorescences.

Understanding how grafts form has fascinated scientists for over a century, and numerous transcription regulators involved in wound healing, callus formation, and grafting have been identified in model plants. However, the genetic architecture underlying grafting traits remains poorly characterized. To address this gap, we aimed to identify quantitative trait loci (QTLs) associated with grafting success rates and grafting-related traits (including callus formation, mechanical strength of the graft union, and post-grafting growth) in a grapevine rootstock mapping population, Vitis berlandieri x V. rupestris. Although no QTLs were identified for grafting success rate, QTLs were identified for component traits revealing a genetic architecture that would have remained hidden when considering grafting success as a single composite phenotype. By integrating QTL and transcriptome data, we identified regulators potentially mediating grafting-related traits. We then showed that one transcription factor that regulates callus formation and grafting in Arabidopsis potentially has a similar role in grapevine by silencing with exogenous double-stranded RNAs and that exogenous application of methyl jasmonate increases callus development at the graft interface. Candidate transcriptional regulators located within QTLs and evidence from gene silencing and hormone treatments suggest the conservation of regulatory mechanisms underlying wound-induced callus formation across plant species.

While the core transcription factor TAF4b is known to regulate meiotic transcription and recombination in model plant Arabidopsis thaliana, its functional specialization and evolutionary dynamics remain poorly understood in paleopolyploid crops. To address this, we investigated how the unique, single-copy TAF4b gene modulates early meiotic progression and reproductive success in Brassica rapa. We characterized multiple independent taf4b TILLING lines for fertility and meiotic behavior, then used this information to select a mutant line for further investigation using a pipeline combining whole-genome background sequencing (DNA-seq), stage-specific anther transcriptomics (RNA-seq), yeast two-hybrid assays, and quantitative cytogenetics. Disruption of the meiosis-enriched TAF4b gene on chromosome A7 universally reduced crossover frequency and depleted HEI10 foci, triggering occasional univalent formation. Global RNA-seq and structural modeling indicate that this phenotype is driven by a precise transcriptional dysregulation and altered molecular interactions restricted to the interphase/leptotene transition. In conclusion, TAF4b acts as a stage-specific upstream gatekeeper of the early prophase I expression program in B. rapa, thereby playing an essential, non-redundant role in its meiotic stability.

Aphids deplete their host plants of amino acids and other essential nutrients by feeding on phloem sap. Autophagy, a conserved process across eukaryotes, maintains cellular homeostasis during starvation and environmental stress by recycling damaged or excess cellular components. Here, we identified and characterized a previously uninvestigated aphid salivary protein, termed Aphid Autophagy Related Factor 1 (AAF1), that is secreted into plant cells and manipulates plant physiology in a manner that benefits the aphid. We show that AAF1 interacts with four known or putative host autophagy related proteins: RABD1 and RABD2a (plant homologs of Ypt1/RAB1), ATG8, a ubiquitin like protein required for autophagosome formation, and PI3PT, a previously uncharacterized ER localized putative lipid binding or lipid transfer protein annotated as a phosphoinositide 3 phosphate transfer protein. We further demonstrate that the RAB binding domain of AAF1 and its interaction with RABD2a may be specifically required for AAF1 function. In addition, we identified unique, highly mobile compartments containing interacting AAF1 and RABD2a that are associated with the endoplasmic reticulum (ER) and dynamically traffic with autophagosomes. We propose that these compartments may be derived from endosomes or endosome to ER contact sites (EERCSs) and serve as platforms for autophagosome trafficking and/or biogenesis. We further propose that AAF1, RABD2a, and PI3PT function together to regulate the formation or function of these compartments, thereby supporting autophagosome formation and trafficking. Finally, we show that stable expression of AAF1 in plants enhances growth and biomass in an autophagy dependent manner.

Lipolytic enzymes play critical roles in plant development, growth, and stress responses. In oat (Avena sativa), they are of particular interest because lipid hydrolysis contributes to rancidity and reduced grain quality, yet little is known about genomic variation in oat lipolytic enzymes. Here, we investigated genomic and expression diversity of the GDSL-esterase/lipase gene family using pangenome, transcriptome, and genome-wide association analyses. Across 22 A. sativa and two A. sterilis genome assemblies, we identified 7,775 GDSL genes (~324 per assembly), revealing wide conservation of ancestral orthologs alongside extensive copy-number variation among homoeologs and paralogs. Transcriptome profiling across 20 genotypes and six tissues demonstrated preferential expression in embryo and root, with tissue-specific patterns highlighting candidates for specific functions, such as seed lipid metabolism. Associations with structural variants further uncovered candidate mutations underlying expression variation among accessions. Comparisons of cultivated and wild oat detected loci with signatures of breeding-based selection, suggesting that GDSL genes have contributed to diversification of the cultivated gene pool. Using 629 whole-genome sequenced accessions, we also identified associations between GDSL regions and agronomic traits, particularly plant height and effective tiller number. Several regions showed patterns consistent with selection, highlighting candidate loci for further investigation of agronomic variation. Together, our results reveal substantial genomic and transcriptional diversity within the oat GDSL gene family and identify candidate genes and genomic regions for improving seed quality and agronomic performance. More broadly, our study demonstrates how pangenomic resources can be used to investigate gene-family diversity and function in complex polyploid crops.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

With a sub-3-micrometer cell and a 27-Mbp diploid genome, the small but mighty Picochlorum celeri doubles within three hours and exceeds 30 g m-2 d-1 outdoors with CO2 supplied. In climate-simulating photobioreactors, productivity declined from 39 g m-2 d-1 under 2.25% CO2 to near zero in air and resumed within hours of CO2 restoration with photosystem II photochemistry largely retained. We profiled the transcriptome, proteome, phosphoproteome, ubiquitinome, acetylome, metabolome and lipidome, including the first site-resolved green-algal ubiquitinome. In air, the Rubisco large subunit tripled to 7.6% of protein while its two small-subunit isoforms, 70% identical, exchanged near-reciprocally, a novel CO2-dependent switch that we propose retunes Rubisco to low CO2. Photorespiratory enzymes increased in abundance whereas proteins involved in external nitrogen assimilation decreased, consistent with increased reliance on internal nitrogen recycling as carbon fixation became limiting. Transcript and protein responses were nearly uncoupled, with cytosolic ribosomal proteins declining despite increased transcripts and plastid ribosomal proteins spared. Phosphorylation was extensively remodeled, as were the lysine modifications ubiquitination, acetylation and CO2-dependent carbamylation, where a CO2-competition assay identified 86 bicarbonate-protected lysines, including RbcL K252. CO2 limitation therefore imposes a reversible growth arrest that reduces cytosolic translational investment while preserving photosynthetic capacity for rapid recovery.

Precise quantitative and spatial control of plant gene expression is typically attributed to cis-regulatory elements in promoters and other non-coding sequences. The extent to which exon-encoded regulatory information shapes plant gene expression and how remains unclear. Here we used BRASSINOSTEROID INSENSITIVE 1 (BRI1) as a model locus and found that it contains an exonic enhancer (EE), which is essential to establish the spatial BRI1 expression pattern. Its disruption restricts BRI1 expression and impairs BRI1-dependent root growth, linking exon-encoded regulatory information and development. The BRI1 EE acts in concert with its promoter, and related receptor kinase genes likewise harbor regulatory information within their coding sequences. Genome-wide surveys show that candidate EEs are associated with highly connected chromatin interaction networks and transcriptionally active loci. Moreover, species comparisons suggest that common features of exon-encoded regulatory information extend across diverse plant lineages. Thus, exon-encoded regulatory information is a vital component of plant gene regulation.

Protein import across the chloroplast outer membrane is mediated by the TOC complex and regulated by GTP hydrolysis at two receptor GTPases, large and small. The former are the {beta}-barrel-containing Toc159 family of proteins (Sun and Jarvis 2023; H. Li and Chiu 2010; Shi and Theg 2013a; Kessler and Schnell 2009). The smallest member of this family in higher plants is Toc90, which has long been regarded as functionally unimportant due to the absence of obvious phenotypes in early mutant studies (Kubis et al. 2004; Infanger et al. 2011a). It was hypothesised that Toc90 is a relic of an ancestral Toc159 protein from early green lineage organisms (Hofmann and Theg 2003; Reumann et al. 2005). Here, we challenge this view by combining genetic, biochemical, phylogenetic, and in silico structural analyses to show that Toc90 is not an ancient remnant, but rather a neofunctionalised derivative of Toc159, and likely the most recently evolved member of the family. We show that Toc90 physically associates with Toc33 as a part of a photosynthetic TOC complex. Furthermore, we identify Toc90 as a regulator of chloroplast ageing. Our findings redefine Toc90 as an active and specialised component of the chloroplast protein import machinery, playing a pivotal role in senescence, especially in reducing photosynthetic protein abundance. Together, these results highlight Toc90 as a promising target for manipulating chloroplast longevity and plant senescence, with potential to improve crop productivity and yield.