Evolution of Thylakoid Structural Diversity.

Oxygenic photosynthesis evolved billions of years ago, becoming Earth's main source of biologically available carbon and atmospheric oxygen. Since then, phototrophic organisms have diversified from prokaryotic cyanobacteria into several distinct clades of eukaryotic algae and plants through endosymbiosis events. This diversity can be seen in the thylakoid membranes, complex networks of lipids, proteins, and pigments that perform the light-dependent reactions of photosynthesis. In this review, we highlight the structural diversity of thylakoids, following the evolutionary history of phototrophic species. We begin with a molecular inventory of different thylakoid components and then illustrate how these building blocks are integrated to form membrane networks with diverse architectures. We conclude with an outlook on understanding how thylakoids remodel their architecture and molecular organization during dynamic processes such as biogenesis, repair, and environmental adaptation.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

Liquid-Liquid Phase Transition Drives Intra-chloroplast Cargo Sorting.

In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.

Oldest thylakoids in fossil cells directly evidence oxygenic photosynthesis.

Today oxygenic photosynthesis is unique to cyanobacteria and their plastid relatives within eukaryotes. Although its origin before the Great Oxidation Event is still debated1-4, the accumulation of O2 profoundly modified the redox chemistry of the Earth and the evolution of the biosphere, including complex life. Understanding the diversification of cyanobacteria is thus crucial to grasping the coevolution of our planet and life, but their early fossil record remains ambiguous5. Extant cyanobacteria include the thylakoid-less Gloeobacter-like group and the remainder of cyanobacteria that acquired thylakoid membranes6,7. The timing of this divergence is indirectly estimated at between 2.7 and 2.0 billion years ago (Ga) based on molecular clocks and phylogenies8-11 and inferred from the earliest undisputed fossil record of Eoentophysalis belcherensis, a 2.018-1.854 Ga pleurocapsalean cyanobacterium preserved in silicified stromatolites12,13. Here we report the oldest direct evidence of thylakoid membranes in a parallel-to-contorted arrangement within the enigmatic cylindrical microfossils Navifusa majensis from the McDermott Formation, Tawallah Group, Australia (1.78-1.73 Ga), and in a parietal arrangement in specimens from the Grassy Bay Formation, Shaler Supergroup, Canada (1.01-0.9 Ga). This discovery extends their fossil record by at least 1.2 Ga and provides a minimum age for the divergence of thylakoid-bearing cyanobacteria at roughly 1.75 Ga. It allows the unambiguous identification of early oxygenic photosynthesizers and a new redox proxy for probing early Earth ecosystems, highlighting the importance of examining the ultrastructure of fossil cells to decipher their palaeobiology and early evolution.

Structure, biogenesis, and evolution of thylakoid membranes.

Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes (TMs) that convert sunlight into chemical energy. These membranes house PSII and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, TMs have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas TMs in cyanobacteria are relatively simple, they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of TM architectures in phototrophs and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes.

CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1) is a photosystem I assembly factor in Arabidopsis.

Photosystem I (PSI) forms a large macromolecular complex of ∼580 kDa that resides in the thylakoid membrane and mediates photosynthetic electron transfer. PSI is composed of 18 protein subunits and nearly 200 co-factors. The assembly of the complex in thylakoid membranes requires high spatial and temporal coordination, and is critically dependent on a sophisticated assembly machinery. Here, we report and characterize CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1), a PSI assembly factor in Arabidopsis (Arabidopsis thaliana). The CEPA1 gene was identified bioinformatically as being co-expressed with known PSI assembly factors. Disruption of the CEPA1 gene leads to a pale phenotype and retarded plant development but does not entirely abolish photoautotrophy. Biophysical and biochemical analyses revealed that the phenotype is caused by a specific defect in PSI accumulation. We further show that CEPA1 acts at the post-translational level and co-localizes with PSI in nonappressed thylakoid membranes. In native gels, CEPA1 co-migrates with thylakoid protein complexes, including putative PSI assembly intermediates. Finally, protein-protein interaction assays suggest cooperation of CEPA1 with the PSI assembly factor PHOTOSYSTEM I ASSEMBLY3 (PSA3). Together, our data support an important but nonessential role of CEPA1 in PSI assembly.

Structure, function, and assembly of PSI in thylakoid membranes of vascular plants.

The photosynthetic apparatus is formed by thylakoid membrane-embedded multiprotein complexes that carry out linear electron transport in oxygenic photosynthesis. The machinery is largely conserved from cyanobacteria to land plants, and structure and function of the protein complexes involved are relatively well studied. By contrast, how the machinery is assembled in thylakoid membranes remains poorly understood. The complexes participating in photosynthetic electron transfer are composed of many proteins, pigments, and redox-active cofactors, whose temporally and spatially highly coordinated incorporation is essential to build functional mature complexes. Several proteins, jointly referred to as assembly factors, engage in the biogenesis of these complexes to bring the components together in a step-wise manner, in the right order and time. In this review, we focus on the biogenesis of the terminal protein supercomplex of the photosynthetic electron transport chain, PSI, in vascular plants. We summarize our current knowledge of the assembly process and the factors involved and describe the challenges associated with resolving the assembly pathway in molecular detail.

The biogenesis and maintenance of PSII: Recent advances and current challenges.

The growth of plants, algae, and cyanobacteria relies on the catalytic activity of the oxygen-evolving PSII complex, which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for PSI in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.

RESISTANCE TO PHYTOPHTHORA1 promotes cytochrome b559 formation during early photosystem II biogenesis in Arabidopsis.

As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in the protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of reactive oxygen species under photoinhibitory-light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Membrane association of active genes organizes the chloroplast nucleoid structure.

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

MFP1 defines the subchloroplast location of starch granule initiation.

Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants "mis"-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, "re" localizing MFP1 to the stromal face of the chloroplast's inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

Chlorophyll to zeaxanthin energy transfer in nonphotochemical quenching: An exciton annihilation-free transient absorption study.

Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, nonphotochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton-exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including the wild type and three NPQ mutants (npq1, npq4, and lut2) generated by CRISPR/Cas9 mutagenesis. The results show a strong correlation between excitation energy transfer from excited Chl Qy to Zea S1 and the xanthophyll cycle during qE activation. Notably, a Lut S1 signal is absent in the npq1 thylakoids which lack zeaxanthin. Additionally, the fifth-order response analysis shows a reduction in the exciton diffusion length (LD) from 62 ± 6 nm to 43 ± 3 nm under high light illumination, consistent with the reduced range of exciton motion being a key aspect of plants' response to excess light.

CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly, photosynthesis, and chloroplast development in Arabidopsis.

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CFO) and a soluble coupling factor (CF1). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF1-CFO assembly. In plants that lacked AtCGL160N, CF1-CFO content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF1 subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF1-ß subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF1 subcomplex, which is critical for the modulation of CF1-CFO activity and photosynthesis.

Arabidopsis Sec14 proteins (SFH5 and SFH7) mediate interorganelle transport of phosphatidic acid and regulate chloroplast development.

Lipids establish the specialized thylakoid membrane of chloroplast in eukaryotic photosynthetic organisms, while the molecular basis of lipid transfer from other organelles to chloroplast remains further elucidation. Here we revealed the structural basis of Arabidopsis Sec14 homology proteins AtSFH5 and AtSFH7 in transferring phosphatidic acid (PA) from endoplasmic reticulum (ER) to chloroplast, and whose function in regulating the lipid composition of chloroplast and thylakoid development. AtSFH5 and AtSFH7 localize at both ER and chloroplast, whose deficiency resulted in an abnormal chloroplast structure and a decreased thickness of stacked thylakoid membranes. We demonstrated that AtSFH5, but not yeast and human Sec14 proteins, could specifically recognize and transfer PA in vitro. Crystal structures of the AtSFH5-Sec14 domain in complex with L-α-phosphatidic acid (L-α-PA) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) revealed that two PA ligands nestled in the central cavity with different configurations, elucidating the specific binding mode of PA to AtSFH5, different from the reported phosphatidylethanolamine (PE)/phosphatidylcholine (PC)/phosphatidylinositol (PI) binding modes. Quantitative lipidomic analysis of chloroplast lipids showed that PA and monogalactosyldiacylglycerol (MGDG), particularly the C18 fatty acids at sn-2 position in MGDG were significantly decreased, indicating a disrupted ER-to-plastid (chloroplast) lipid transfer, under deficiency of AtSFH5 and AtSFH7. Our studies identified the role and elucidated the structural basis of plant SFH proteins in transferring PA between organelles, and suggested a model for ER-chloroplast interorganelle phospholipid transport from inherent ER to chloroplast derived from endosymbiosis of a cyanobacteriumproviding a mechanism involved in the adaptive evolution of cellular plastids.

Proton gradient across the chloroplast thylakoid membrane governs the redox regulatory function of ATP synthase.

Chloroplast ATP synthase (CFoCF1) synthesizes ATP by using a proton electrochemical gradient across the thylakoid membrane, termed ΔµH+, as an energy source. This gradient is necessary not only for ATP synthesis but also for reductive activation of CFoCF1 by thioredoxin, using reducing equivalents produced by the photosynthetic electron transport chain. ΔµH+ comprises two thermodynamic components: pH differences across the membrane (ΔpH) and the transmembrane electrical potential (ΔΨ). In chloroplasts, the ratio of these two components in ΔµH+ is crucial for efficient solar energy utilization. However, the specific contribution of each component to the reductive activation of CFoCF1 remains unclear. In this study, an in vitro assay system for evaluating thioredoxin-mediated CFoCF1 reduction is established, allowing manipulation of ΔµH+ components in isolated thylakoid membranes using specific chemicals. Our biochemical analyses revealed that ΔpH formation is essential for thioredoxin-mediated CFoCF1 reduction on the thylakoid membrane, whereas ΔΨ formation is nonessential.

The outstanding capacity of Prasiola antarctica to thrive in contrasting harsh environments relies on the constitutive protection of thylakoids and on morphological plasticity.

The determination of physiological tolerance ranges of photosynthetic species and of the biochemical mechanisms underneath are fundamental to identify target processes and metabolites that will inspire enhanced plant management and production for the future. In this context, the terrestrial green algae within the genus Prasiola represent ideal models due to their success in harsh environments (polar tundras) and their extraordinary ecological plasticity. Here we focus on the outstanding Prasiola antarctica and compare two natural populations living in very contrasting microenvironments in Antarctica: the dry sandy substrate of a beach and the rocky bed of an ephemeral freshwater stream. Specifically, we assessed their photosynthetic performance at different temperatures, reporting for the first time gnsd values in algae and changes in thylakoid metabolites in response to extreme desiccation. Stream population showed lower α-tocopherol content and thicker cell walls and thus, lower gnsd and photosynthesis. Both populations had high temperatures for optimal photosynthesis (around +20°C) and strong constitutive tolerance to freezing and desiccation. This tolerance seems to be related to the high constitutive levels of xanthophylls and of the cylindrical lipids di- and tri-galactosyldiacylglycerol in thylakoids, very likely related to the effective protection and stability of membranes. Overall, P. antarctica shows a complex battery of constitutive and plastic protective mechanisms that enable it to thrive under harsh conditions and to acclimate to very contrasting microenvironments, respectively. Some of these anatomical and biochemical adaptations may partially limit photosynthesis, but this has a great potential to rise in a context of increasing temperature.

2-Cys peroxiredoxins contribute to thylakoid lipid unsaturation by affecting ω-3 fatty acid desaturase 8.

Fatty acid unsaturation levels affect chloroplast function and plant acclimation to environmental cues. However, the regulatory mechanism(s) controlling fatty acid unsaturation in thylakoid lipids is poorly understood. Here, we have investigated the connection between chloroplast redox homeostasis and lipid metabolism by focusing on 2-Cys peroxiredoxins (Prxs), which play a central role in balancing the redox state within the organelle. The chloroplast redox network relies on NADPH-dependent thioredoxin reductase C (NTRC), which controls the redox balance of 2-Cys Prxs to maintain the reductive activity of redox-regulated enzymes. Our results show that Arabidopsis (Arabidopsis thaliana) mutants deficient in 2-Cys Prxs contain decreased levels of trienoic fatty acids, mainly in chloroplast lipids, indicating that these enzymes contribute to thylakoid membrane lipids unsaturation. This function of 2-Cys Prxs is independent of NTRC, the main reductant of these enzymes, hence 2-Cys Prxs operates beyond the classic chloroplast regulatory redox system. Moreover, the effect of 2-Cys Prxs on lipid metabolism is primarily exerted through the prokaryotic pathway of glycerolipid biosynthesis and fatty acid desaturase 8 (FAD8). While 2-Cys Prxs and FAD8 interact in leaf membranes as components of a large protein complex, the levels of FAD8 were markedly decreased when FAD8 is overexpressed in 2-Cys Prxs-deficient mutant backgrounds. These findings reveal a function for 2-Cys Prxs, possibly acting as a scaffold protein, affecting the unsaturation degree of chloroplast membranes.

Genetically manipulated chloroplast stromal phosphate levels alter photosynthetic efficiency.

The concentration of inorganic phosphate (Pi) in the chloroplast stroma must be maintained within narrow limits to sustain photosynthesis and to direct the partitioning of fixed carbon. However, it is unknown if these limits or the underlying contributions of different chloroplastic Pi transporters vary throughout the photoperiod or between chloroplasts in different leaf tissues. To address these questions, we applied live Pi imaging to Arabidopsis (Arabidopsis thaliana) wild-type plants and 2 loss-of-function transporter mutants: triose phosphate/phosphate translocator (tpt), phosphate transporter 2;1 (pht2;1), and tpt pht2;1. Our analyses revealed that stromal Pi varies spatially and temporally, and that TPT and PHT2;1 contribute to Pi import with overlapping tissue specificities. Further, the series of progressively diminished steady-state stromal Pi levels in these mutants provided the means to examine the effects of Pi on photosynthetic efficiency without imposing nutritional deprivation. ΦPSII and nonphotochemical quenching (NPQ) correlated with stromal Pi levels. However, the proton efflux activity of the ATP synthase (gH+) and the thylakoid proton motive force (pmf) were unaltered under growth conditions, but were suppressed transiently after a dark to light transition with return to wild-type levels within 2 min. These results argue against a simple substrate-level limitation of ATP synthase by depletion of stromal Pi, favoring more integrated regulatory models, which include rapid acclimation of thylakoid ATP synthase activity to reduced Pi levels.

Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance.

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.