Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Plastid DNA is a major source of nuclear genome complexity and of RNA genes in the orphan crop moringa.

BACKGROUND: Unlike Transposable Elements (TEs) and gene/genome duplication, the role of the so-called nuclear plastid DNA sequences (NUPTs) in shaping the evolution of genome architecture and function remains poorly studied. We investigate here the functional and evolutionary fate of NUPTs in the orphan crop Moringa oleifera (moringa), featured by the highest fraction of plastid DNA found so far in any plant genome, focusing on (i) any potential biases in their distribution in relation to specific nuclear genomic features, (ii) their contribution to the emergence of new genes and gene regions, and (iii) their impact on the expression of target nuclear genes. RESULTS: In agreement with their potential mutagenic effect, NUPTs are underrepresented among structural genes, although their overall transcription levels and broadness were only lower when involved exonic regions; the occurrence of plastid DNA generally did not result in a broader expression, except among those affected in introns by older NUPTs. In contrast, we found a strong enrichment of NUPTs among specific superfamilies of retrotransposons and several classes of RNA genes, including those participating in the protein biosynthetic machinery (i.e., rRNA and tRNA genes) and a specific class of regulatory RNAs. A significant fraction of NUPT RNA genes was found to be functionally expressed, thus potentially contributing to the nuclear pool. CONCLUSIONS: Our results complete our view of the molecular factors driving the evolution of nuclear genome architecture and function, and support plastid DNA in moringa as a major source of (i) genome complexity and (ii) the nuclear pool of RNA genes.

Comparative and phylogenetic analyses of Loranthaceae plastomes provide insights into the evolutionary trajectories of plastome degradation in hemiparasitic plants.

BACKGROUND: The lifestyle transition from autotrophy to heterotrophy often leads to extensive degradation of plastomes in parasitic plants, while the evolutionary trajectories of plastome degradation associated with parasitism in hemiparasitic plants remain poorly understood. In this study, phylogeny-oriented comparative analyses were conducted to investigate whether obligate Loranthaceae stem-parasites experienced higher degrees of plastome degradation than closely related facultative root-parasites and to explore the potential evolutionary events that triggered the 'domino effect' in plastome degradation of hemiparasitic plants. RESULTS: Through phylogeny-oriented comparative analyses, the results indicate that Loranthaceae hemiparasites have undergone varying degrees of plastome degradation as they evolved towards a heterotrophic lifestyle. Compared to closely related facultative root-parasites, all obligate stem-parasites exhibited an elevated degree plastome degradation, characterized by increased downsizing, gene loss, and pseudogenization, thereby providing empirical evidence supporting the theoretical expectation that evolution from facultative parasitism to obligate parasitism may result in a higher degree of plastome degradation in hemiparasites. Along with infra-familial divergence in Loranthaceae, several lineage-specific gene loss/pseudogenization events occurred at deep nodes, whereas further independent gene loss/pseudogenization events were observed in shallow branches. CONCLUSIONS: The findings suggest that in addition to the increasing levels of nutritional reliance on host plants, cladogenesis can be considered as another pivotal evolutionary event triggering the 'domino effect' in plastome degradation of hemiparasitic plants. These findings provide new insights into the evolutionary trajectory of plastome degradation in hemiparasitic plants.

Complete organelle genomes of Korean fir, Abies koreana and phylogenomics of the gymnosperm genus Abies using nuclear and cytoplasmic DNA sequence data.

Abies koreana E.H.Wilson is an endangered evergreen coniferous tree that is native to high altitudes in South Korea and susceptible to the effects of climate change. Hybridization and reticulate evolution have been reported in the genus; therefore, multigene datasets from nuclear and cytoplasmic genomes are needed to better understand its evolutionary history. Using the Illumina NovaSeq 6000 and Oxford Nanopore Technologies (ONT) PromethION platforms, we generated complete mitochondrial (1,174,803 bp) and plastid (121,341 bp) genomes from A. koreana. The mitochondrial genome is highly dynamic, transitioning from cis- to trans-splicing and breaking conserved gene clusters. In the plastome, the ONT reads revealed two structural conformations of A. koreana. The short inverted repeats (1186 bp) of the A. koreana plastome are associated with different structural types. Transcriptomic sequencing revealed 1356 sites of C-to-U RNA editing in the 41 mitochondrial genes. Using A. koreana as a reference, we additionally produced nuclear and organelle genomic sequences from eight Abies species and generated multiple datasets for maximum likelihood and network analyses. Three sections (Balsamea, Momi, and Pseudopicea) were well grouped in the nuclear phylogeny, but the phylogenomic relationships showed conflicting signals in the mitochondrial and plastid genomes, indicating a complicated evolutionary history that may have included introgressive hybridization. The obtained data illustrate that phylogenomic analyses based on sequences from differently inherited organelle genomes have resulted in conflicting trees. Organelle capture, organelle genome recombination, and incomplete lineage sorting in an ancestral heteroplasmic individual can contribute to phylogenomic discordance. We provide strong support for the relationships within Abies and new insights into the phylogenomic complexity of this genus.

Transcriptome analysis reveals molecular mechanisms underlying chloroplast biogenesis in albino Agave angustifolia plantlets.

Albino plants display partial or complete loss of photosynthetic pigments and defective thylakoid membrane development, consequently impairing plastid function and development. These distinctive attributes render albino plants excellent models for investigating chloroplast biogenesis. Despite their potential, limited exploration has been conducted regarding the molecular alterations underlying these phenotypes, extending beyond photosynthetic metabolism. In this study, we present a novel de novo transcriptome assembly of an albino somaclonal variant of Agave angustifolia Haw., which spontaneously emerged during the micropropagation of green plantlets. Additionally, RT-qPCR analysis was employed to validate the expression of genes associated with chloroplast biogenesis, and plastome copy numbers were quantified. This research aims to gain insight into the molecular disruptions affecting chloroplast development and ascertain whether the expression of critical genes involved in plastid development and differentiation is compromised in albino tissues of A. angustifolia. Our transcriptomic findings suggest that albino Agave plastids exhibit high proliferation, activation of the protein import machinery, altered transcription directed by PEP and NEP, dysregulation of plastome expression genes, reduced expression of photosynthesis-associated nuclear genes, disruption in the tetrapyrrole and carotenoid biosynthesis pathway, alterations in the plastid ribosome, and an increased number of plastome copies, among other alterations.

Plastid phylogenomics and fossil evidence provide new insights into the evolutionary complexity of the 'woody clade' in Saxifragales.

BACKGROUND: The "woody clade" in Saxifragales (WCS), encompassing four woody families (Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae), is a phylogenetically recalcitrant node in the angiosperm tree of life, as the interfamilial relationships of the WCS remain contentious. Based on a comprehensive sampling of WCS genera, this study aims to recover a robust maternal backbone phylogeny of the WCS by analyzing plastid genome (plastome) sequence data using Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods, and to explore the possible causes of the phylogenetic recalcitrance with respect to deep relationships within the WCS, in combination with molecular and fossil evidence. RESULTS: Although the four WCS families were identically resolved as monophyletic, the MP analysis recovered different tree topologies for the relationships among Altingiaceae, Cercidiphyllaceae, and Daphniphyllaceae from the ML and BI phylogenies. The fossil-calibrated plastome phylogeny showed that the WCS underwent a rapid divergence of crown groups in the early Cretaceous (between 104.79 and 100.23 Ma), leading to the origin of the stem lineage ancestors of Altingiaceae, Cercidiphyllaceae, Daphniphyllaceae, and Hamamelidaceae within a very short time span (∼4.56 Ma). Compared with the tree topology recovered in a previous study based on nuclear genome data, cytonuclear discordance regarding the interfamilial relationships of the WCS was detected. CONCLUSIONS: Molecular and fossil evidence imply that the early divergence of the WCS might have experienced radiative diversification of crown groups, extensive extinctions at the genus and species levels around the Cretaceous/Paleocene boundary, and ancient hybridization. Such evolutionarily complex events may introduce biases in topological estimations within the WCS due to incomplete lineage sorting, cytonuclear discordance, and long-branch attraction, potentially impacting the accurate reconstruction of deep relationships.

Investigation of heterotrophs reveals new insights in dinoflagellate evolution.

Dinoflagellates are diverse and ecologically important protists characterized by many morphological and molecular traits that set them apart from other eukaryotes. These features include, but are not limited to, massive genomes organized using bacterially-derived histone-like proteins (HLPs) and dinoflagellate viral nucleoproteins (DVNP) rather than histones, and a complex history of photobiology with many independent losses of photosynthesis, numerous cases of serial secondary and tertiary plastid gains, and the presence of horizontally acquired bacterial rhodopsins and type II RuBisCo. Elucidating how this all evolved depends on knowing the phylogenetic relationships between dinoflagellate lineages. Half of these species are heterotrophic, but existing molecular data is strongly biased toward the photosynthetic dinoflagellates due to their amenability to cultivation and prevalence in culture collections. These biases make it impossible to interpret the evolution of photosynthesis, but may also affect phylogenetic inferences that impact our understanding of character evolution. Here, we address this problem by isolating individual cells from the Salish Sea and using single cell, culture-free transcriptomics to expand molecular data for dinoflagellates to include 27 more heterotrophic taxa, resulting in a roughly balanced representation. Using these data, we performed a comprehensive search for proteins involved in chromatin packaging, plastid function, and photoactivity across all dinoflagellates. These searches reveal that 1) photosynthesis was lost at least 21 times, 2) two known types of HLP were horizontally acquired around the same time rather than sequentially as previously thought; 3) multiple rhodopsins are present across the dinoflagellates, acquired multiple times from different donors; 4) kleptoplastic species have nucleus-encoded genes for proteins targeted to their temporary plastids and they are derived from multiple lineages, and 5) warnowiids are the only heterotrophs that retain a whole photosystem, although some photosynthesis-related electron transport genes are widely retained in heterotrophs, likely as part of the iron-sulfur cluster pathway that persists in non-photosynthetic plastids.

Complete plastid genome of Iris orchioides and comparative analysis with 19 Iris plastomes.

Iris is a cosmopolitan genus comprising approximately 280 species distributed throughout the Northern Hemisphere. Although Iris is the most diverse group in the Iridaceae, the number of taxa is debatable owing to various taxonomic issues. Plastid genomes have been widely used for phylogenetic research in plants; however, only limited number of plastid DNA markers are available for phylogenetic study of the Iris. To understand the genomic features of plastids within the genus, including its structural and genetic variation, we newly sequenced and analyzed the complete plastid genome of I. orchioides and compared it with those of 19 other Iris taxa. Potential plastid markers for phylogenetic research were identified by computing the sequence divergence and phylogenetic informativeness. We then tested the utility of the markers with the phylogenies inferred from the markers and whole-plastome data. The average size of the plastid genome was 152,926 bp, and the overall genomic content and organization were nearly identical among the 20 Iris taxa, except for minor variations in the inverted repeats. We identified 10 highly informative regions (matK, ndhF, rpoC2, ycf1, ycf2, rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI and psbK-trnQ) and inferred a phylogeny from each region individually, as well as from their concatenated data. Remarkably, the phylogeny reconstructed from the concatenated data comprising three selected regions (rpoC2, ycf1 and ycf2) exhibited the highest congruence with the phylogeny derived from the entire plastome dataset. The result suggests that this subset of data could serve as a viable alternative to the complete plastome data, especially for molecular diagnoses among closely related Iris taxa, and at a lower cost.

Exploring Plastomic Resources in Sempervivum (Crassulaceae): Implications for Phylogenetics.

The plastid organelle is vital for photosynthesis and energy production. Advances in sequencing technology have enabled the exploration of plastomic resources, offering insights into plant evolution, diversity, and conservation. As an important group of horticultural ornamentals in the Crassulaceae family, Sempervivum plants are known for their unique rosette-like structures and reproduction through offsets. Despite their popularity, the classification status of Sempervivum remains uncertain, with only a single plastome sequence currently available. Furthermore, codon usage bias (CUB) is a widespread phenomenon of the unbalanced usage of synonymous codons in the coding sequence (CDS). However, due to the limited available plastid data, there has been no research that focused on the CUB analysis among Sempervivum until now. To address these gaps, we sequenced and released the plastomes of seven species and one subspecies from Sempervivum, revealing several consistent patterns. These included a shared 110 bp extension of the rps19 gene, 14 hypervariable regions (HVRs) with distinct nucleotide diversity (π: 0.01173 to 0.02702), and evidence of selective pressures shaping codon usage. Notably, phylogenetic analysis robustly divided the monophyletic clade into two sections: Jovibarba and Sempervivum. In conclusion, this comprehensive plastomic resource provides valuable insights into Sempervivum evolution and offers potential molecular markers for DNA barcoding.

First Record of Comparative Plastid Genome Analysis and Phylogenetic Relationships among Corylopsis Siebold & Zucc. (Hamamelidaceae).

Corylopsis Siebold & Zucc. (Hamamelidaceae) is widely used as a horticultural plant and comprises approximately 25 species in East Asia. Molecular research is essential to distinguish Corylopsis species, which are morphologically similar. Molecular research has been conducted using a small number of genes but not in Corylopsis. Plastid genomes of Corylopsis species (Corylopsis gotoana, Corylopsis pauciflora, and Corylopsis sinensis) were sequenced using next-generation sequencing techniques. Repeats and nucleotide diversity that could be used as DNA markers were also investigated. A phylogenetic investigation was carried out using 79 protein-coding genes to infer the evolutionary relationships within the genus Corylopsis. By including new plastomes, the overall plastid genome structure of Corylopsis was similar. Simple sequence repeats of 73-106 SSRs were identified in the protein-coding genes of the plastid genomes, and 33-40 long repeat sequences were identified in the plastomes. The Pi value of the rpl33_rps18 region, an intergenic spacer, was the highest. Phylogenetic analysis demonstrated that Corylopsis is a monophyletic group and Loropetalum is closely related to Corylopsis. C. pauciflora, C. gotoana, and C. spicata formed a clade distributed in Japan, whereas C. sinensis, C. glandulifera, and C. velutina formed a clade that was distributed in China.

New plastids, old proteins: repeated endosymbiotic acquisitions in kareniacean dinoflagellates.

Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.

Phylogenomics reveals Adeleorina are an ancient and distinct subgroup of Apicomplexa.

Apicomplexans are a diverse phylum of unicellular eukaryotes that share obligate relationships with terrestrial and aquatic animal hosts. Many well-studied apicomplexans are responsible for several deadly zoonotic and human diseases, most notably malaria caused by Plasmodium. Interest in the evolutionary origin of apicomplexans has also spurred recent work on other more deeply-branching lineages, especially gregarines and sister groups like squirmids and chrompodellids. But a full picture of apicomplexan evolution is still lacking several lineages, and one major, diverse lineage that is notably absent is the adeleorinids. Adeleorina apicomplexans comprises hundreds of described species that infect invertebrate and vertebrate hosts across the globe. Although historically considered coccidians, phylogenetic trees based on limited data have shown conflicting branch positions for this subgroup, leaving this question unresolved. Phylogenomic trees and large-scale analyses comparing cellular functions and metabolism between major subgroups of apicomplexans have not incorporated Adeleorina because only a handful of molecular markers and a couple organellar genomes are available, ultimately excluding this group from contributing to our understanding of apicomplexan evolution and biology. To address this gap, we have generated complete genomes from mitochondria and plastids, as well as multiple deep-coverage single-cell transcriptomes of nuclear genes from two Adeleorina species, Klossia helicina and Legerella nova, and inferred a 206-protein phylogenomic tree of Apicomplexa. We observed distinct structures reported in species descriptions as remnant host structures surrounding adeleorinid oocysts. Klossia helicina and L. nova branched, as expected, with monoxenous adeleorinids within the Adeleorina and their mitochondrial and plastid genomes exhibited similarity to published organellar adeleorinid genomes. We show with a phylogeneomic tree and subsequent phylogenomic analyses that Adeleorina are not closely related to any of the currently sampled apicomplexan subgroups, and instead fall as a sister to a large clade encompassing Coccidia, Protococcidia, Hematozoa, and Nephromycida, collectively. This resolves Adeleorina as a key independently-branching group, separate from coccidians, on the tree of Apicomplexa, which now has all known major lineages sampled.

Complex Endosymbioses I: From Primary to Complex Plastids, Serial Endosymbiotic Events.

A considerable part of the diversity of eukaryotic phototrophs consists of algae with plastids that evolved from endosymbioses between two eukaryotes. These complex plastids are characterized by a high number of envelope membranes (more than two) and some of them contain a residual nucleus of the endosymbiotic alga called a nucleomorph. Complex plastid-bearing algae are thus chimeric cell assemblies, eukaryotic symbionts living in a eukaryotic host. In contrast, the primary plastids of the Archaeplastida (plants, green algae, red algae, and glaucophytes) possibly evolved from a single endosymbiosis with a cyanobacterium and are surrounded by two membranes. Complex plastids have been acquired several times by unrelated groups of eukaryotic heterotrophic hosts, suggesting that complex plastids are somewhat easier to obtain than primary plastids. Evidence suggests that complex plastids arose twice independently in the green lineage (euglenophytes and chlorarachniophytes) through secondary endosymbiosis, and four times in the red lineage, first through secondary endosymbiosis in cryptophytes, then by higher-order events in stramenopiles, alveolates, and haptophytes. Engulfment of primary and complex plastid-containing algae by eukaryotic hosts (secondary, tertiary, and higher-order endosymbioses) is also responsible for numerous plastid replacements in dinoflagellates. Plastid endosymbiosis is accompanied by massive gene transfer from the endosymbiont to the host nucleus and cell adaptation of both endosymbiotic partners, which is related to the trophic switch to phototrophy and loss of autonomy of the endosymbiont. Such a process is essential for the metabolic integration and division control of the endosymbiont in the host. Although photosynthesis is the main advantage of acquiring plastids, loss of photosynthesis often occurs in algae with complex plastids. This chapter summarizes the essential knowledge of the acquisition, evolution, and function of complex plastids.

Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

Functional conservation of GENOMES UNCOUPLED1 in plastid-to-nucleus retrograde signaling in tomato.

Retrograde signaling between plastids and the nucleus is vital for chloroplast biogenesis and environmental responses. GENOMES UNCOUPLED1 (GUN1) was proposed to be a central integrator of multiple retrograde signaling pathways in the model plant Arabidopsis thaliana (Arabidopsis). However, the function of GUN1 orthologs in other plant species has not been well studied. Here, we found that many GUN1 orthologs from the Solanaceae family have a short N-terminus before the first pentatricopeptide repeat (PPR) motif which is predicted as intrinsically disordered regions (IDRs). Functional analyses of tomato (Solanum lycopersicum L.) GUN1 (SlGUN1), which does not contain N-terminal IDRs, show that it can complement the GUN phenotype of the Arabidopsis gun1 mutant (Atgun1). However, in contrast to the AtGUN1 protein, which does contain the N-terminal IDRs, the SlGUN1 protein is highly accumulated even after chloroplast biogenesis is completed, suggesting that the N-terminal IDRs may determine the stability of the GUN1 protein. Furthermore, we generated tomato Slgun1 genome-edited mutants via the CRISPR-Cas9 system. The Slgun1 mutants exhibited a typical GUN phenotype under lincomycin (Lin) or norflurazon (NF) treatment. Moreover, Slgun1 mutants are hypersensitive to low concentrations of Lin or NF. Taken together, our results suggest that, although lacking the N-terminal IDRs, SlGUN1 plays conserved roles in plastid retrograde signaling in tomato plants.

Multiple factors interact in editing of PPR-E+-targeted sites in maize mitochondria and plastids.

RNA cytidine-to-uridine editing is essential for plant organellar gene expression. Pentatricopeptide repeat (PPR)-E+ proteins have been proposed to bind to target sites and recruit the cytidine deaminase AtDYW2, facilitated by AtNUWA. Here we analyze the function of ZmNUWA, ZmDYW2A, and ZmDYW2B and their relationships with other editing factors in maize. The zmdyw2a and zmdyw2b single mutants are normal, but the zmdyw2a::zmdyw2b and zmnuwa mutants are severely arrested in seed development. ZmNUWA, ZmDYW2A, and ZmDYW2B are dual localized in mitochondria and plastids. Loss of ZmNUWA decreases the editing at 99 mitochondrial sites and 8 plastid sites. Surprisingly, loss of ZmDYW2A:ZmDYW2B affects almost the same set of sites targeted by PPR-E+ proteins. ZmNUWA interacts with ZmDYW2A and ZmDYW2B, suggesting that ZmNUWA recruits ZmDYW2A/2B in the editing of PPR-E+-targeted sites in maize. Further protein interaction analyses show that ZmNUWA and ZmDYW2A/2B interact with ZmMORF1, ZmMORF8, ZmMORF2, and ZmMORF9 and that ZmOZ1 interacts with ZmORRM1, ZmDYW2A, ZmDYW2B, ZmMORF8, and ZmMORF9. These results suggest that the maize mitochondrial PPR-E+ editosome contains PPR-E+, ZmDYW2A/2B, ZmNUWA, and ZmMORF1/8, whereas the plastid PPR-E+ editosome is composed of PPR-E+, ZmDYW2A/2B, ZmNUWA, ZmMORF2/8/9, ZmORRM1, and ZmOZ1.

Phylogenomics and plastomics offer new evolutionary perspectives on Kalanchoideae (Crassulaceae).

BACKGROUND AND AIMS: Kalanchoideae is one of three subfamilies within Crassulaceae and contains four genera. Despite previous efforts, the phylogeny of Kalanchoideae remains inadequately resolved with persistent issues including low support, unstructured topologies and polytomies. This study aimed to address two central objectives: (1) resolving the pending phylogenetic questions within Kalanchoideae by using organelle-scale 'barcodes' (plastomes) and nuclear data; and (2) investigating interspecific diversity patterns among Kalanchoideae plastomes. METHODS: To explore the plastome evolution in Kalanchoideae, we newly sequenced 38 plastomes representing all four constituent genera (Adromischus, Cotyledon, Kalanchoe and Tylecodon). We performed comparative analyses of plastomic features, including GC and gene contents, gene distributions at the IR (inverted repeat) boundaries, nucleotide divergence, plastomic tRNA (pttRNA) structures and codon aversions. Additionally, phylogenetic inferences were inferred using both the plastomic dataset (79 genes) and nuclear dataset (1054 genes). KEY RESULTS: Significant heterogeneities were observed in plastome lengths among Kalanchoideae, strongly correlated with LSC (large single copy) lengths. Informative diversities existed in the gene content at SSC/IRa (small single copy/inverted repeat a), with unique patterns individually identified in Adromischus leucophyllus and one major Kalanchoe clade. The ycf1 gene was assessed as a shared hypervariable region among all four genera, containing nine lineage-specific indels. Three pttRNAs exhibited unique structures specific to Kalanchoideae and the genera Adromischus and Kalanchoe. Moreover, 24 coding sequences revealed a total of 41 lineage-specific unused codons across all four constituent genera. The phyloplastomic inferences clearly depicted internal branching patterns in Kalanchoideae. Most notably, by both plastid- and nuclear-based phylogenies, our research offers the first evidence that Kalanchoe section Eukalanchoe is not monophyletic. CONCLUSIONS: This study conducted comprehensive analyses on 38 newly reported Kalanchoideae plastomes. Importantly, our results not only reconstructed well-resolved phylogenies within Kalanchoideae, but also identified highly informative unique markers at the subfamily, genus and species levels. These findings significantly enhance our understanding of the evolutionary history of Kalanchoideae.

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.