Hans-Henning Kunz

Plant metabolism heavily relies on plastids, derived from once free-living organisms. However, how two distinct organisms merged into one remains currently only partially understood. Protein-mediated metabolite exchange across the double-membrane plastid envelope is essential for plant cell function. Here, we investigate the evolutionary origins of plastid outer envelope proteins (OEPs) involved in these transport processes. The mosaic nature of the nuclear genome and the deep evolutionary distance since plastid acquisition are major challenges. To address them, we combine sequence-based analyses with emerging structure-based tools, which together enable more sensitive evolutionary comparisons than traditional methods alone. To uncover distinct evolutionary trajectories, we focused on 27 OEPs from metabolite transport and protein import, with diverse structures. We found that the β-barrel proteins involved in metabolite transport were recruited in a stepwise manner and structural homologs of some OEPs point to early recruitment via endosymbiotic gene transfer (EGT). Protein import is sequence-wise conserved across Archaeplastida, although refinements occur across Chloroplastdia and structural turnover is implicated. Notably, OEP40 shows a recent structural rearrangement, lacking clear structural homologs in plants other than Arabidopsis, yet retaining sequence conservation across all major land-plant lineages. The JASSY-like family is found across plastid-bearing species, while true JASSY orthologs emerged in embryophytes likely via a stable recruitment of the characteristic lipid-binding domain. Overall, our findings highlight the dynamic nature of the plastid outer envelope and show how new functions could evolve through structural reshaping and novel domain recruitment.

Summary: NPQ relaxation is thought to limit crop biomass and yield under natural, fluctuating light. The thylakoid K⁺/H⁺ exchanger KEA3 accelerates NPQ relaxation by lowering the lumen proton concentration and thus should affect biomass and yield under frequent light fluctuations. To investigate the impact of KEA3-dependent NPQ relaxation on Solanum lycopersicum (tomato), we generated knock-out and over-expression lines and measured NPQ, gas exchange, biomass and yield. We constructed a mathematical model to compare species-specific NPQ relaxation dynamics in tomato and the model plant Arabidopsis thaliana . In polytunnel and controlled fluctuating-light conditions, KEA3-mediated NPQ relaxation did not translate into consistent differences in tomato biomass or yield. Although KEA3 levels strongly affected NPQ relaxation, more so than in A. thaliana , the resulting effects on CO ₂ assimilation were minor. Modeling results indicated that, besides minor changes in KEA3 regulation, the activity of PsbS, the primary NPQ trigger, largely determines the species-specific differences in NPQ relaxation. Structural models from both species predict sequence variation with impact on PsbS activation. Manipulating KEA3 levels in tomato did not link NPQ relaxation with productivity. However, the results support a robust cross-species mathematical model that accurately predicts the molecular interactions governing NPQ dynamics.

Summary: Plant cell walls constitute dynamic barriers that are essential for defense against pathogens. Beyond a structural role in blocking pathogen infection, innate mechanisms monitor cell wall integrity (CWI) in Arabidopsis thaliana and trigger local responses where integrity is impaired. Here, we show that THESEUS1 (THE1)‐dependent CWI signaling induces accumulation of the antimicrobial metabolite camalexin upon cell wall damage (CWD) caused by isoxaben (ISX)‐induced cellulose biosynthesis inhibition or infection with the phytopathogenic fungus Colletotrichum higginsianum . CWD caused by ISX alters THE1 plasma membrane nanodomain organization and involves calcium signaling components that modulate camalexin production. Induction of camalexin requires jasmonic acid (JA) signaling and JA‐dependent expression of the transcription factor MYB47. In line with its antagonistic function on CWI signaling, the plant elicitor peptide Pep3 suppresses camalexin biosynthesis downstream of THE1 by inhibiting JA‐dependent pathways. Our findings reveal a regulatory network where CWI and Pep3 signaling modulate antimicrobial defense via JA‐mediated camalexin production. This network requires input from independent CWD‐induced pathways, providing insights into how plants balance distinct defense processes in response to cell wall stress.

Plants constantly encounter adverse environmental interactions. One organelle is particularly specialized in stress signaling and phytohormone synthesis: the plastid. Calcium (Ca ²⁺ ), a key second messenger, is known to intersect with cellular phytohormone signaling networks. While cytosolic Ca ²⁺ dynamics have been studied extensively, the physiological relevance of stromal Ca ²⁺ transients and the identity of channels mediating rapid Ca ²⁺ flux into plastids remain largely unexplored. In this study, we provide evidence for PLASTID ENVELOPE ION CHANNELS (PECs) as long-sought mediators of fast-activating cation channel-like currents. We show that PEC expression is jasmonic acid (JA)-induced to augment stromal Ca ²⁺ transients under stress. Loss of PECs results in decreased JA priming and failure to elicit full defense responses after wounding. In turn, PEC1 overexpression improves Botrytis cinerea tolerance. Our findings link stromal Ca ²⁺ signaling with JA-dependent stress responses and position PECs as modulators of plant defense.

Members of the K⁺ efflux antiporter (KEA) family fulfill key roles in plastids and the endomembrane system. Plants and green algae possess at least one KEA mediating K⁺/H⁺ exchange across the plastid inner envelope (IE) membrane. Recently, IE KEAs were shown to be essential for plastid gene expression (PGE), chloroplast development, and photosynthesis. Plants lacking these antiporters exhibit reduced stromal protein synthesis and accumulation of unprocessed rRNA precursors. KEA proteins comprise a conserved monovalent cation/proton antiporter 2 (CPA2) domain and a regulatory K⁺ transport and NAD-binding (KTN) domain. IE KEAs are distinguished by an additional ∼500-amino-acid N-terminal extension containing a coiled-coil (CC) domain embedded within a largely intrinsically disordered region (IDR). Intrigued by this unusual architecture, we performed phylogenetic analyses, revealing that this N-terminal fusion arose early and has been conserved throughout the green lineage. We then investigated the oligomeric state, native distribution, and function of the N-terminal domain. Using Arabidopsis thaliana , we found that IE KEAs localize to discrete clusters within the inner envelope membrane and assemble into complexes of approximately 600 kDa. Finally, complementary approaches using a functional KEA1 variant lacking the core N-terminal domains (KEA1ΔN) indicate that this extension plays a regulatory rather than an essential role. Our findings uncover an evolutionarily ancient regulatory module that shapes the molecular organization and function of IE KEAs, advancing our understanding of plastid ion and pH homeostasis and plastid ribosome integrity. One-sentence summary: Plastid KEA1/2 proteins feature a unique N-terminal extension that modulates potassium transport activity in a yet unknown manner but is not essential for normal plant growth under ambient conditions.

In Arabidopsis thaliana, seamless plastid gene expression and development depend on finely balanced ion homeostasis across the inner envelope (IE) membrane, maintained by the K+/H+ antiporters AtKEA1/2. To assess whether these functions are retained across mono- and polyplastidic representatives of the green lineage, we studied CrKEA1, the sole IE KEA homolog in the unicellular alga Chlamydomonas reinhardtii. Using CRISPR/Cas9, we generated a Cr-kea1 knockout mutant that exhibits impaired photoautotrophic growth, chloroplast deformation, and photoinhibition. Transcriptomics revealed strong induction of ribosome biogenesis genes and reduced abundance of transcripts associated with cell and plastid division. Further RNA analyses confirmed defects in stromal rRNA maturation of Cr-kea1, paralleling observations from Arabidopsis kea1kea2 mutants. Expression of CrKEA1 in Arabidopsis rescued growth and rRNA maturation in At-kea1kea2, demonstrating functional continuity after the ancient divergence between the 2 lineages. Cross-species transcriptomic comparisons further revealed that IE KEA loss elicits both shared and species-specific transcriptional responses: PhANG repression was conserved between algae and plants, whereas activation of the chloroplast unfolded protein response (cpUPR) and reduced expression of genes tied to cell-cycle and plastid fission occurred only in Chlamydomonas. Single-cell time-lapse imaging confirmed that Cr-kea1 exhibits an increased frequency of aberrant cytokinesis, unequal division, and division failure. Our findings demonstrate that while IE KEA transporters fulfill conserved roles in maintaining the conditions for plastid gene expression, their integration into broader cellular networks has diverged between unicellular chlorophytes and embryophytes (land plants). This underscores a lineage-dependent tuning of plastid-nucleus communication shaped by organismal complexity and plastid number.

In bacteria, protein mistranslation can improve stress tolerance. Mitochondria and plastids evolved from bacteria and use a prokaryotic-type expression machinery to synthesize proteins. Interestingly, fungi and animal mitochondria are highly sensitive to mistranslation, which for instance manifests in lethal mitochondrial cardiomyopathy disorder. The response in plant cells is unknown. Glutaminyl-transfer RNAs (Gln-tRNA ^Gln ) of bacteria and endosymbiotic organelles are synthesized indirectly. Initially, tRNA ^Gln is aminoacylated with glutamate. Subsequently, Gln is produced through trans-amidation by the aminoacyl-tRNA amido-transferase complex GatCAB. Consequentially, compromised GatCAB activity yields misloaded Glu-tRNA ^Gln . Arabidopsis mutants with decreased GatCAB levels provide global insights into organellar mistranslation in plants: Our proteomics analyses revealed mutant-specific high plastid and low mitochondrial Gln-to-Glu misincorporation rates in organellar-expressed protein complexes with only modest protein abundance changes in plastids and none in mitochondria. We identify efficient compensatory mechanisms that mitigate the physiological consequences of elevated mistranslation in mutants. Interestingly, wild-type plants under temperature stress also have altered Gln-to-Glu misincorporation while temperature acclimation differs in Gln-to-Glu hypermistranslating mutants. Our study indicates that the response toward organellar mistranslation varies among eukaryotes and enables future detailed investigation of mistranslation compensation mechanisms in plant cells.

Plant cell biochemistry is highly compartmentalized. Hence, forward genetic screening in an organelle-specific manner holds great potential to reveal novel loci controlling cell physiology. However, conventional forward genetics is a) limited by functional redundancy that can mask phenotypes in plant gene families and b) lacks organelle specificity. Here, we present the plastid-focused artificial microRNA ( pamiR ) resource designed to silence individual nuclear genes or co-targetable gene-family subsets encoding the organelle’s proteome. The library contains 2,285 artificial microRNAs directed against 2,202 genes selected from a plastid-enriched, proteomics-supported target set. The constructs are expressed by a constitutive UBQ10 promoter in a binary vector carrying the FAST seed-fluorescence marker, allowing transformant selection before germination. Two proof-of-concept screens, one using photosynthetic phenotyping and one using paclobutrazol, a gibberellin biosynthesis inhibitor, recovered lines with phenotypes previously associated with simultaneous perturbation of multiple members of redundant plastid gene families such as light-harvesting complex b1 (LHCB1), K ⁺ efflux antiporter (KEA), and 9-cis-epoxycarotenoid dioxygenase (NCED). We experimentally confirmed specific target depletion for selected candidates, including paralogue-resolved LHCB1 depletion by mass spectrometry, and provide predicted off-target annotations for all 2,285 guides. Untargeted LHCB family members remained unchanged, and across the proteins included in the proteome-wide differential-abundance analysis, no additional significantly decreased protein was detected. KEA1 immunoreactive signal also fell below the immunoblot detection limit in pamiRkea1/2 and an independent KEA1/KEA2 amiRNA line. Together, these data establish pamiR as a plastid-specific screening resource and provide a framework for prioritizing and independently validating candidate phenotypes. One-sentence summary: The pamiR library enables family-level silencing of nuclear genes encoding plastid proteins and provides per-construct target and predicted off-target annotations for organelle-focused forward genetic screens.

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this acclimation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigated the role of Chloroplast Lipid Remodeling Protein 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and increases in the galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrated that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses revealed broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation.