Markus Schwarzländer

Malate is a central metabolite in plant energy metabolism and biosynthesis and a major carrier of carbon and reducing equivalents between chloroplasts, the cytosol, and mitochondria. Here, we combined reverse genetics with multiple complementary approaches to investigate how disruption of the major mitochondrial malate-converting enzymes affects plant performance under different environmental conditions. We generated two independent mutant lines (mdh1 me1 me2) lacking the predominant mitochondrial malate dehydrogenase isoform MDH1 and both subunits of NAD-dependent malic enzyme (NAD-ME), ME1 and ME2. Growth under contrasting photoperiods and irradiances revealed a conditional phenotype that was most pronounced under short-day/low-light condition. Under this condition, the mutants exhibited impaired growth and photosynthetic performance, accompanied by cytosolic redox imbalance and altered chloroplast ultrastructure. Transfer experiments demonstrated that this phenotype was reversible and determined by the prevailing growth conditions: plants initially grown under short days and low light recovered in newly formed leaves after transfer to long-day/normal-light condition, whereas the reciprocal transfer reproduced the growth impairment in newly formed leaves. Transcriptomic and proteomic analyses revealed dawn-associated perturbations in photosynthetic and redox homeostasis, characterized by reduced investment in chloroplast translation and photosynthetic machinery alongside enhanced proteome maintenance, photoprotection and repair, and iron/ROS homeostasis. Branched-chain amino acids also accumulated in the mutants at the end of the night under low light. Together, these results show that the combined loss of mitochondrial MDH1 and NAD-ME causes a reversible, light-regime-dependent impairment of Arabidopsis growth and photosynthetic performance, highlighting the importance of the simultaneous contribution of both the MDH1 and NAD-ME systems to mitochondrial malate conversion when photosynthetic energy input is limited.

Cellular energy homeostasis relies on the precise exchange of nucleotides across subcellular compartments. The transport of ADP and ATP is essential for maintaining cellular energy balance. Transporters of these molecules are abundant proteins in the mitochondrial membrane, mediating their exchange between the mitochondrial matrix and the cytosol. In this study, we investigated the function of ADP/ATP Carrier 1 (AAC1) in plants to understand whether its regulation and function are similar to those described in animal and yeast systems. Our results revealed that AAC1 specifically transports ADP and ATP, while aac1 mutants displayed lower O2 consumption without evident decreases in cytosolic ATP levels. However, total adenylate profiling revealed altered adenylate balance and reduced adenylate energy charge in aac1 roots, indicating that reduced AAC1 function perturbs energy homeostasis beyond changes in ATP content alone. AAC1 was also required for proper mitochondrial structure, as aac1 mutant mitochondria exhibited a less electron-dense matrix and occasional absence of cristae. Suppressing AAC1 expression in plants altered vegetative and reproductive growth, reducing leaf number and root elongation while increasing silique size and seed weight. These findings provide novel insight into the role of AAC1 in mitochondrial energy dynamics. Biochemical analyses show a high specificity of AAC1 for adenine nucleotides, facilitating ADP/ATP exchange driven by the mitochondrial membrane potential, while excluding monophosphates and divalent ATP complexes. By correlating these biochemical characteristics with impaired mitochondrial respiration, altered adenylate balance, ultrastructural defects, and altered energy allocation, our study highlights the broader physiological impacts of disrupted adenylate transport mediated by AAC1.

Summary: Seeds enable plant embryos to endure extended periods and environmental extremes. Orthodox seeds acquire desiccation tolerance during maturation, before metabolic quiescence. Coordinated thylakoid disassembly and Chl degradation lead to embryo degreening, a hallmark of orthodox seed maturation. While degreening is hormonally controlled, our understanding of the molecular players involved and its physiological significance remains incomplete. By serendipity, we observed a stay‐green seed phenotype in an Arabidopsis thaliana mutant lacking two LATE EMBRYOGENESIS ABUNDANT (LEA) proteins, LEA42 and LEA48, which we identified as the most abundant LEA proteins in seed mitochondria and plastids. Using lea48xlea42 as a model for impaired LEA function in seed mitochondria and plastids, proteomic, transcriptomic and transmission electron microscopy analyses indicate incomplete thylakoid degradation. Freshly harvested lea48xlea42 seeds were unaffected in germination but were impaired in germination after high‐temperature/high‐humidity ageing. Genetic complementation with specifically targeted LEA expression in either mitochondria or chloroplasts revealed that seed longevity support is associated with plastidial, but not mitochondrial, LEA. Light exposure of mature seeds (‘light storage’) reduced longevity, providing a physiological framework for embryo degreening. Our data identify a role for LEA proteins in remodelling seed plastids before maturation drying and highlight LEA‐dependent embryo degreening as a decisive contributor to seed longevity.

SUMMARY: Land plant mitochondria and plastids are endosymbiotic organelles that possess their own, reduced genomes. Gene expression is largely regulated at the transcript level, predominantly by nuclear‐encoded proteins. Notably, up to thousands of cytidines in organellar RNAs are specifically edited to uridines, mostly restoring conserved genetic information. Key players in this process are nuclear‐encoded pentatricopeptide repeat (PPR) proteins, which bind target RNAs in a one‐PPR:one‐nucleotide manner, following a two‐amino‐acid‐based code. Cytidine deamination is catalyzed by intrinsic or trans ‐ acting DYW domains. The modularity and programmability of PPR‐RNA interactions make PPR editing factors attractive candidates for the development of biotechnological tools. However, RNA target selection is not determined by the ‘PPR code’ alone, hampering the generation of highly effective and specific editors. Refining such tools requires efficient assays to dissect the determinants of editor‐RNA interaction. Here, we introduce a rapid assay to assess PPR editor specificity through transient expression in the cytosol of Nicotiana benthamiana , which offers a large and diverse transcriptome that enables extensive off‐target editing. Profiling of these off‐targets provides comprehensive information on an editor's target selectivity. Compared with a previous setup in the model moss Physcomitrium patens , which relies on stable transformation, the required experimental time is reduced from months to days. We demonstrate the power of the system by characterizing the off‐targets of the moss editor PpPPR56. The new assay allows rapid functional assessment of editing factor variants and removes a bottleneck in understanding editor‐RNA interactions, which is a prerequisite for the rational improvement of PPR‐based RNA editing tools.

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

Current dogma holds that CO ₂ fixation by photosynthesis requires additional ATP production via cyclic electron transfer, mediated by two distinct pathways involving the NADH dehydrogenase-like complex (NDH) and proton gradient regulation 5 protein (PGR5), to augment the ATP relative to NADPH produced by linear electron transfer. Here we use the MgATP ²⁻ fluorescent biosensor ATeam1.03-nD/nA and find that, while proton motive force in the Arabidopsis pgr5CAS and pgr5 ndho mutants is only 50–75% that of the wild type, chloroplast MgATP ²⁻ levels are unchanged. These data demonstrate that disequilibrium exists between the proton motive force and MgATP ²⁻ concentration in vivo, indicating that cyclic electron transfer is not required for ATP augmentation.

SUMMARY: Plants have evolved elaborate acclimation strategies to withstand adverse environments, involving all levels of their function and organization. While macroscopic movements through differential growth that enable the physical re‐shaping and ‐positioning of organs have been a major field of study, the highly active dynamics of cell organelles within the immobile plant cell hold major unresolved questions. Here, we argue that the precise positioning of cell organelles is not only critical for cell division and development but also represents a fundamental principle in regulating cellular metabolism and underpinning effective responses to biotic and abiotic stresses. Although the positioning of individual organelles, such as nuclei and chloroplasts, and probably to a lesser extent, mitochondria, peroxisomes, endoplasmic reticulum tubules, Golgi bodies, and lipid droplets, is clearly controlled by developmental and environmental stimuli, the underlying mechanisms and functional significance remain unclear in many instances. We discuss steps required to improve our understanding of how intracellular positioning of organelles in general, and of chloroplasts and mitochondria in particular, contributes to metabolic regulation. Drawing on the recent discovery that three glycolytic enzymes can physically tether mitochondria and chloroplasts, we highlight approaches and concepts to establish how the spatial arrangement of organelles relative to one another underpins regulation of metabolism and stress responses.

All organisms fuel and build themselves through their energy metabolism. While classic biochemistry conceptualizes the fluxes of energy and matter, our understanding of how energy metabolism works in vivo contains major gaps. One reason is that energy metabolism spans multiple scales, from enzymes to organs, and shifts dynamically at environmental and developmental transitions, resulting in a degree of complexity that is presently impossible to capture. Genetically encoded fluorescent biosensors have started to bridge several critical gaps by enabling live monitoring of metabolites across scales. Recently, several paradigms of energy metabolism have started to shift, driven by the expansion of biosensing tools. This review explores advancements in our understanding of plant energy metabolism driven by fluorescent protein biosensing, highlights emerging concepts and open questions, and discusses how available tools, and much-needed future innovations, can unlock the potential of biosensing toward understanding in vivo plant energy metabolism and its effective modification.

Understanding plant molecular responses to flooding is crucial for strategies to increase resilience. Plants respond to submergence-induced low oxygen (hypoxia) through decreased plant cysteine oxidase (PCO) activity, which stabilizes group VII ethylene response factors (ERFVIIs), master regulators of metabolic and anatomic acclimation responses ^1–4 . Rapid reoxygenation on desubmergence induces a burst of reactive oxygen species (ROS) generation and metabolic reconfiguration ^5,6 ; however, how plants mitigate this post-hypoxic stress to facilitate submergence recovery has remained unknown. Here we report that ERFVIIs are also important in post-submergence recovery, remaining stable upon reoxygenation through ROS-mediated PCO inhibition. Stabilized ERFVIIs are retained at hypoxia-responsive promoters, becoming repressors of typical hypoxia marker genes but upregulators of genes involved in ROS homeostasis and oxidative stress protection. Our findings suggest that PCOs and ERFVIIs integrate signals from both oxygen and ROS to coordinate ERFVII stability through submergence-induced hypoxia and desubmergence stress to promote plant survival and recovery.

T-DNA insertion mutants are widely used to disrupt genes and infer their functions, yet the insertions can also trigger unintended genomic changes that confound phenotypic interpretation. Here, we used T-DNA insertion mutants affecting the major mitochondrial malate dehydrogenase (MDH1) and the heterodimeric NAD-dependent malic enzymes (ME1 and ME2) to examine their functional coordination across photoperiods and irradiance regimes. Under short days, especially at low light intensity, mdh1xme2 mutants were markedly smaller than wild type and, unexpectedly, than the mdh1xme1xme2 triple mutant, and they showed a more pronounced reduction in photosynthetic capacity. ME1 was undetectable in mdh1xme2 , implying that the double and triple mutants effectively lack heterodimeric ME and should therefore behave similarly, contrary to what we observed. Whole-genome analysis resolved this discrepancy by revealing that the MDH1 T-DNA insertion in mdh1xme2 is accompanied by a major rearrangement, a 137-kbp duplication downstream of the insertion site, which was absent in the mdh1xme1xme2 triple mutant. This duplication increased gene dosage and elevated transcript abundance across the duplicated interval, while proteomics detected 5 of the 38 encoded proteins, including PEPC1. mdh1xme2 accumulated oxaloacetate-derived amino acids and displayed an altered carbon/nitrogen balance, making PEPC1 a plausible contributor to the exacerbated mdh1xme2 phenotype. Together, our data indicate that a T-DNA-linked structural variant can amplify expression of dozens of genes and intensify phenotypes at specific conditions, thereby affecting the interpretation of genotype-phenotype relationships. Because Agrobacterium -mediated DNA transfer also underpins many genome-editing workflows, our findings argue that structural validation around insertion/editing loci should be considered essential when interpreting T-DNA-derived plant lines.